ISMARLEY LAGE HORTA MORAIS

TRATAMENTO BIOLÓGICO DE EFLUENTES DE FÁBRICAS DE POLPA

CELULÓSICA E PAPEL COM LODO AERÓBIO GRANULAR

Tese apresentada à Universidade

Federal de Viçosa, como parte das
exigências do Programa de Pós-
Graduação em Engenharia Civil, para
obtenção do título de Doctor
Scientiae.

VIÇOSA
MINAS GERAIS – BRASIL
2016
Ficha catalográfica preparada pela Biblioteca Central da Universidade
Federal de Viçosa - Câmpus Viçosa

T
Morais, Ismarley Lage Horta, 1984-
M827t Tratamento biológico de efluentes de fábricas de polpa
2016 celulósica e papel com lodo aeróbio granular / Ismarley Lage
Horta Morais. – Viçosa, MG, 2016.
vii, 82f : il. (algumas color.) ; 29 cm.

Inclui anexos.
Orientador: Cláudio Mudadu Silva.
Tese (doutorado) - Universidade Federal de Viçosa.
Inclui bibliografia.

1. Águas residuais - Purificação - Tratamento biológico.

2. Lodo aeróbio granular. 3. Celulose e papel. I. Universidade
Federal de Viçosa. Departamento de Engenharia Civil. Programa
de Pós-graduação em Engenharia Civil. II. Título.

CDD 22. ed. 628.3

 ISMARLEY LAGE HORTA MORAIS

TRATAMENTO BIOLÓGICO DE EFLUENTES DE FÁBRICAS DE POLPA

CELULÓSICA E PAPEL COM LODO AERÓBIO GRANULAR

Tese apresentada à Universidade

Federal de Viçosa, como parte das
exigências do Programa de Pós-
Graduação em Engenharia Civil, para
obtenção do título de Doctor
Scientiae.

APROVADA: 15 de dezembro de 2016.

Ana Augusta Passos Rezende André Pereira Rosa

Ann Honor Mounteer Cláudio Arcanjo de Sousa

(Coorientadora)

Cláudio Mudadu Silva

(Orientador)
 AGRADECIMENTOS

A Universidade Federal de Viçosa, ao Departamento de Engenharia Civil e ao

Laboratório de Celulose e Papel pela oportunidade de realização do curso.

Aos meus pais, Ismar e Isabel, e minhas irmãs por me apoiarem em todas as
minhas decisões e torcerem sempre pelo meu sucesso. Vocês são importantes demais para
mim.

A Natália pela companhia, apoio e carinho durante todo esse tempo. Só tenho a
agradecer por estar sempre presente e nunca me deixar desanimar.

Ao professor Claudio Mudadu Silva, pela orientação e ensinamentos durante tanto

tempo. Foram muitos anos de amizade e trabalho juntos nos quais aprendi e cresci muito.
Tenho muito a agradecer pelo apoio, sobretudo na etapa de finalização dessa tese.

As professoras Ann Honor Mounteer e Ana Augusta que também acompanharam

e contribuíram muito para minha formação acadêmica desde a graduação, além das
contribuições importantes neste trabalho.

Aos professores e colegas André Pereira Rosa e Cláudio Arcanjo de Sousa pela
convivência em Viçosa e por aceitarem contribuir com o trabalho.

A todos do Laboratório de Meio Ambiente do LCP pela companhia e os bons

momentos de convivência, especialmente à Iolanda e Raquel pela ajuda no
monitoramento dos reatores e realização do experimento. Aos colegas dos laboratórios
de química da madeira, polpação, branqueamento e papel pelas conversas nos corredores,
sala de estudantes e cantina do LCP, sobretudo à Sandra e Larisse que me toleraram esses
anos e a quem eu sempre recorria para pedir emprestado um reagente ou equipamento
para realização das análises.

Ao CNPq e CAPES pelo suporte financeiro concedido.

A CENIBRA pelo fornecimento dos efluentes utilizados nos reatores em batelada.

Ao engenheiro Felipe Cristelli que se dispôs a ajudar e sempre me recebeu na fábrica para
as coletas dos efluentes, além dos funcionários da ETB que ajudaram a coletar as
amostras.

A todas as pessoas que contribuíram para que eu realizasse este trabalho.

ii
 BIOGRAFIA

Ismarley Lage Horta Morais, filho de Ismar de Assis Morais e Isabel Lage
Moreira, nasceu na cidade de Timóteo, Minas Gerais, em 8 de novembro de 1984.

Em março de 2004 iniciou o curso de Engenharia Ambiental na Universidade

Federal de Viçosa, Minas Gerais, graduando-se em janeiro de 2009.

Em março de 2009, iniciou o curso de pós-graduação em Ciência Florestal, na

área de ambiência, na Universidade Federal de Viçosa desenvolvendo trabalhos na área
de tratamento aeróbio granular de efluentes de máquina de papel e obteve o título de
Magister Scientiae em fevereiro de 2011.

Começou a lecionar disciplinas de matemática e física para os cursos de

engenharia da Unipac de Conselheiro Lafaiete em abril de 2011 e, em novembro de 2012,
começou a trabalhar como engenheiro ambiental na Construtora Século XXI.

Em março de 2014 retornou a Viçosa para iniciar o curso de pós-graduação em

engenharia civil, na área de saneamento e meio ambiente e obteve o título de Doctor
Scientiae em dezembro de 2016.

iii
 ÍNDICE

RESUMO ...................................................................................................................... VI
ABSTRACT ................................................................................................................. VII
I. INTRODUÇÃO GERAL ........................................................................................ 1
CAPÍTULO 1................................................................................................................... 4
RECENT DEVELOPMENTS ON AEROBIC SLUDGE GRANULATION
TECHNOLOGY.............................................................................................................. 4
1. INTRODUCTION ................................................................................................... 4
2. FORMATION OF GRANULAR AEROBIC SLUDGE ...................................... 6
2.1. EXTRACELLULAR POLYMERIC SUBSTANCES ....................................................... 9
2.2. DIVALENT CATIONS ADDITION ......................................................................... 12
2.3. MICROBIAL COAGGREGATION .......................................................................... 15
2.4. IMPORTANT MICROORGANISMS IN GRANULATION PROCESSES .......................... 16
2.5. GRANULE STABILITY ........................................................................................ 19
2.6. NUTRIENT REMOVAL ........................................................................................ 21
2.7. BIODEGRADATION, BIOACCUMULATION AND BIOSORPTION ............................. 23
3. MEMBRANE BIOREACTOR WITH GRANULAR SLUDGE ...................... 24
4. AEROBIC GRANULAR SLUDGE AT HIGH TEMPERATURES ................ 26
5. FULL-SCALE AEROBIC GRANULAR SLUDGE SYSTEMS ...................... 27
6. REFERENCES ...................................................................................................... 29
CAPÍTULO 2................................................................................................................. 38
STABILITY AND RESISTANCE OF AEROBIC GRANULAR SLUDGE IN
BIOREACTORS AFTER ADDITION OF CALCIUM ............................................ 38
ABSTRACT ................................................................................................................... 38
INTRODUCTION ......................................................................................................... 39
METHODOLOGY........................................................................................................ 41
RESULTS AND DISCUSSION ................................................................................... 43
CONCLUSIONS ........................................................................................................... 51
ACKNOWLEDGEMENTS .......................................................................................... 51
REFERENCES .............................................................................................................. 52
CAPÍTULO 3................................................................................................................. 55
IDENTIFICAÇÃO DOS MICRORGANISMOS FAVORÁVEIS À FORMAÇÃO
DE GRÂNULOS AERÓBIOS MESOFÍLICOS PRESENTES NO TRATAMENTO
BIOLÓGICO DE EFLUENTES DE FÁBRICA DE PAPEL ................................... 55
RESUMO ....................................................................................................................... 55
1. INTRODUÇÃO ..................................................................................................... 55
2. METODOLOGIA ................................................................................................. 57
iv
 2.1. OBTENÇÃO DOS GRÂNULOS AERÓBIOS E DOS ISOLADOS .............. 57
2.2. INDICE DE CO-AGREGAÇÃO COM AUSÊNCIA DE UM ISOLADO ...... 58
2.3. EXTRAÇÃO E ANÁLISE DAS SPE ............................................................. 58
2.3.1. Quantificação de carboidratos ................................................................... 58
2.3.2. Quantificação de proteínas e ácidos húmicos ........................................... 59
2.3.3. Determinação de Carbono Orgânico Total (COT) ................................... 59
2.4. IDENTIFICAÇÃO DOS ISOLADOS OBTIDOS NA FORMAÇÃO DOS
GRÂNULOS ............................................................................................................... 59
2.4.1. Extração do DNA ....................................................................................... 59
2.4.2. Amplificação de DNA................................................................................. 60
3. RESULTADOS E DISCUSSÃO .......................................................................... 61
3.1. ENSAIO DE CO-AGREGAÇÃO - DENSIDADE ÓTICA DOS
CONSÓRCIOS ........................................................................................................... 61
3.2. DETERMINAÇÃO DAS SPE ........................................................................ 62
3.2.1. Quantificação de carboidratos ................................................................... 62
3.2.2. Quantificação de Proteínas........................................................................ 63
3.2.3. Quantificação de ácidos húmicos .............................................................. 65
3.3. IDENTIFICAÇÃO DOS ISOLADOS OBTIDOS .......................................... 67
4. CONCLUSÕES ..................................................................................................... 69
5. AGRADECIMENTOS.......................................................................................... 70
6. REFERENCIAS BIBLIOGRÁFICAS ................................................................ 71
II. CONCLUSÕES E RECOMENDAÇÕES GERAIS........................................... 76
ANEXOS ........................................................................................................................ 78
DETERMINAÇÃO DO TAMANHO MÉDIO DOS GRÂNULOS ................................................ 79
DETERMINAÇÃO DA TAXA ESPECÍFICA DE CRESCIMENTO DOS GRÂNULOS .................... 79
DETERMINAÇÃO DA VELOCIDADE MÉDIA DE SEDIMENTAÇÃO ...................................... 79
DETERMINAÇÃO DA RESISTÊNCIA DOS GRÂNULOS ....................................................... 80
OBSERVAÇÕES MICROSCÓPICAS ................................................................................... 81
PURIFICAÇÃO DOS AMPLICONS ..................................................................................... 81

v
 RESUMO
MORAIS, Ismarley Lage Horta, D.Sc., Universidade Federal de Viçosa, dezembro de
2016. Tratamento biológico de efluentes de fábrica de polpa celulósica e papel com
lodo aeróbio granular. Orientador: Cláudio Mudadu Silva. Coorientadora: Ann Honor
Mounteer.

O tratamento de águas residuárias com lodo aeróbio granular apresenta muitas vantagens
em comparação ao processo convencional de lodos ativados com lodo floculento. Os
grânulos são agregados microbianos densos e compactos que possibilitam uma maior
retenção de biomassa no reator biológico e uma elevada capacidade de sedimentação,
favorecendo a remoção biológica de matéria orgânica, nutrientes, compostos tóxicos e
clarificação final do efluente devido à estrutura e propriedade de sedimentação do lodo.
Estes benefícios resultaram em um aumento do interesse de implantação do processo de
tratamento com lodo aeróbio granular e a busca de maiores informações à respeito da
formação, estabilidade e a influência dos parâmetros operacionais sobre a granulação.
Assim, este trabalho apresenta uma revisão bibliográfica com a compilação das
informações recentes sobre o lodo aeróbio granular incluindo a possibilidade de utilização
dos grânulos aeróbios em biorreatores a membrana, em elevadas temperaturas e as
aplicações em plantas de tratamento de larga escala. Foi realizada, ainda, a avaliação da
adição de 100 mg.L-1 e 200mg.L-1 de cálcio na estabilidade, resistência mecânica e
diâmetro dos grânulos formados em reatores em batelada sequencial alimentados com
efluente de uma fábrica de polpa celulósica kraft. Os reatores apresentaram eficiências
similares de remoção de matéria orgânica e o diâmetro médio dos grânulos foi de cerca
de 11 mm em todos os reatores, embora os grânulos formados no reator que recebeu 100
mg.L-1 de Ca2+ apresentou velocidade de sedimentação 36% superior aos demais e maior
resistência mecânica. A melhoria da granulação pode ser obtida ainda pela seleção de
microrganismos que contribuem para a formação dos agregados. A produção de
substâncias poliméricas extracelulares (SPE) pelas bactérias é um dos fatores que
influencia a agregação celular, uma vez que as SPE agem como agente cimentante e
atuam na adesão entre as células. A produção de SPE de dezenove isolados microbianos,
obtidos de grânulos aeróbios formados no tratamento de efluente de fábrica de papel
reciclado foi avaliada e seis isolados dos gêneros Staphylococcus, Agrobacterium,
Enterobacter e Rhodococcus melhoraram a granulação biológica. A ausência destes
isolados nos testes de co-agregação reduziu a relação entre proteínas e polissacarídeos
(relação PN/PS) e diminuiu a formação de agregados.

vi
 ABSTRACT
MORAIS, Ismarley Lage Horta, D.Sc., Universidade Federal de Viçosa, December, 2016.
Biological treatment of pulp and paper mill effluents with aerobic granular sludge.
Adviser: Claudio Mudadu Silva. Co-adviser: Ann Honor Mounteer.

Aerobic granular sludge wastewater treatment has many advantages over the
conventional activated sludge process. The granules are dense and compact microbial
aggregates that allow a higher biomass retention in the biological reactor and a high
settling velocity, favoring the biological removal of organic matter, nutrients, toxic
substances and improves wastewater clarification. Due to the sludge structure and
settleability, these benefits have attracted considerable interest in the implementation of
the aerobic granular sludge process and givenrise to the need for better understanding of
the formation, stability and influence of the operational parameters on the granulation.
Thus, this work was divided into three chapters. Chapter 1 presents a review of recent
developments on aerobic granular sludge including the possibility of using aerobic
granules in membrane bioreactors, at high temperatures and for a full-scale
implementation. The addition of divalent cations in the reactors can enhance granulation
and granule stability. In Chapter 2, the effect of the addition of 100 mg.L-1 and 200 mg.L-
1
of calcium in the stability, mechanical strength and diameter of the granules formed in
sequential batch reactors (SBR) fed with pulp mill effluent was evaluated. The reactors
showed similar organic matter removal efficiencies and granule size was approximately
11 mm in all SBR, although the granules formed in the reactor with addition of 100 mg.L-
1
of Ca2+ had a settling velocity 36% higher and greater mechanical resistance than the
others. Granulation can also be enhanced by the selection of microorganisms that
contribute to the aggregates formation. Bacterial extracellular polymeric substances
(EPS) production is one factor that contributes to cell aggregation, since EPS acts as an
intercellular cement that may reinforce cohesion inside the bacterial clusters. In Chapter
3, EPS production of nineteen microbial isolates obtained from aerobic granules formed
in the recycled paper wastewater treatment was evaluated and six isolates of the genera
Staphylococcus, Agrobacterium, Enterobacter and Rhodococcus contributed to
biological granulation. The absence of these isolates in the co-aggregation tests reduced
the protein-polysaccharide ratio (PN / PS ratio) and reduced the aggregates formation.

vii
 I. Introdução geral
O sistema de lodos ativados é um dos processos de tratamento biológico de
efluentes mais utilizados mundialmente em fábricas de polpa celulósica e papel. No
processo, microrganismos são responsáveis pela oxidação da matéria orgânica do efluente
em reatores aeróbios e, em seguida, o lodo biológico é separado do efluente tratado por
um processo de sedimentação em decantadores secundários. O lodo sedimentado nos
decantadores retorna aos reatores de forma a manter uma alta concentração de
microrganismos capazes de realizar a degradação da matéria orgânica.

A eficiência do processo de lodos ativados depende da capacidade de

sedimentação do lodo biológico. Os flocos biológicos de baixa densidade, que possuem
reduzida velocidade de sedimentação, não são retidos pelo decantador e deterioram a
qualidade do efluente tratado, mesmo que a oxidação da matéria orgânica tenha sido
realizada eficientemente no reator.

O arraste de lodo junto ao efluente tratado no decantador secundário de processos

de lodos ativados é um problema típico nas estações de tratamento de efluentes (ETE). O
indesejado arraste de lodo pode ter causas diversas. A ocorrência de variações na
quantidade e qualidade do efluente de alimentação que podem provocar choques de carga
orgânica e de vazão; a presença de compostos tóxicos à microbiota; a variação de pH e
temperatura; a falta de oxigênio e nutrientes são fatores que podem afetar negativamente
a formação dos flocos e provocar a perda de lodo no decantador secundário. As
propriedades de sedimentação de lodo são frequentemente consideradas como limitantes
da eficiência do processo (YE et al., 2016).

A mudança de morfologia dos agregados microbianos de flocos (lodo floculento

convencional) para grânulos (lodo aeróbio granular) nos reatores aeróbios ocorre a partir
da alteração de alguns parâmetros de operação dos processos de lodos ativados tais como
a redução do tempo de decantação e o aumento da aeração. Os grânulos são agregados
densos e de tamanho maior do que os flocos. Grânulos aeróbios apresentam uma maior
velocidade de sedimentação e uma maior resistência a compostos tóxicos do que os flocos
convencionais. Esse fato tem sido explicado pela sua estrutura compacta e à matriz de
substâncias poliméricas extracelulares (SPE) presentes nos grânulos (WAN et al., 2015).

A utilização de biorreatores aeróbios com lodo granular é incipiente no mundo.

Desde a sua descoberta em 1991 (LEE et al., 2010; SARMA; TAY; CHU, 2016), esse
tema tem sido objeto de várias pesquisas que buscam viabilizar o processo uma vez que

1
possui muitas vantagens sobre o processo convencional de lodo floculento. A literatura
tem apresentado trabalhos conduzidos majoritariamente em laboratório e com efluentes
sintéticos. Existe um número reduzido de trabalhos com efluentes reais, sobretudo
provenientes da produção de polpa celulósica e papel (HONGLEI et al., 2013; LIU;
NGUYEN; PAICE, 2010; MORAIS; SILVA; BORGES, 2016).

A busca de maiores conhecimentos nessa área tem sido intensa por se tratar de um
tema inovador e a compilação desses conhecimentos em forma de uma revisão
bibliográfica é desejável.

A formação e a estabilidade dos grânulos formados são fatores importantes para a

aplicação do tratamento de efluentes utilizando o lodo aeróbio granular. A presença de
pelo menos uma fase com pouca disponibilidade de substrato, um reduzido tempo de
sedimentação e a presença de cátions metálicos divalentes são responsáveis pelo êxito da
granulação aeróbia (HUANG et al., 2012)

A adição de cátions como o cálcio neutraliza a carga negativa presente na

superfície da célula bacteriana e aumenta a agregação do lodo. Os cátions provocam um
aumento da força iônica e reduzem a espessura da camada dupla ao redor da célula,
levando a diminuição da força repulsiva (DING et al., 2015). Além disso, precipitados de
cálcio incorporados ao lodo como sais de carbonato e fosfato também favorecem a
sedimentação (YE et al., 2016). Não se sabe ainda em que concentrações o cálcio
beneficia ou prejudica a formação do lodo aeróbio granular. A maioria dos trabalhos, com
concentrações entre 100 e 200 mg.L-1, mostrou efeito positivo da adição do cálcio,
enquanto aquelas acima de 300 mg.L-1 formaram precipitados de cálcio e causaram
problemas na granulação biológica (YU; TAY; FANG, 2001). Além disso, muitos
estudos avaliaram o crescimento de microrganismos de cultura pura (HAO et al., 2016).
É necessário examinar a influência dos íons metálicos sobre a fixação microbiana e
formação dos grânulos aeróbios, sobretudo para efluentes industriais.

As SPE são polímeros complexos de elevado peso molecular e constituem os

principais componentes dos bioflocos e biofilmes (WANG et al., 2015). As SPE contêm
grande quantidade de compostos como proteínas, polissacarídeos, ácidos húmicos, ácidos
nucléicos e fosfolipídeos, responsáveis pelo armazenamento de energia e nutrientes, além
da resistência ao ataque de fungos e substâncias tóxicas (YAN et al., 2015). São
responsáveis, ainda, por mudanças na sedimentabilidade, na hidrofobicidade da superfície
do lodo e influenciam na formação do lodo granular (YAN et al., 2015).

2
 A formação de grânulos pode ser obtida pela seleção de culturas microbianas que
possibilitem a diminuição do tempo de formação dos grânulos, o aumento da densidade
e a manutenção da capacidade de degradação dos mesmos (IVANOV et al., 2006).
Grânulos mais densos são formados devido a interações específicas envolvendo as SPE
da matriz do grânulo (CAUDAN et al., 2014).

A primeira parte da tese trata-se de uma revisão bibliográfica sobre granulação

aeróbia com foco nos fatores que influenciam a formação de grânulos, o seu
desenvolvimento e sua estabilidade. São também apresentadas as possibilidades de
utilização de grânulos aeróbios em biorreatores a membrana (BRM), em elevadas
temperaturas ou para a remoção de nutrientes e compostos tóxicos, além de aplicações do
lodo aeróbio granular em reatores de larga escala. A segunda parte é a avaliação da
influência da adição de cálcio na granulação em relação à eficiência de remoção de
matéria orgânica, ao aumento da velocidade de sedimentação e resistência mecânica dos
grânulos e a composição das SPE. A terceira parte é a avaliação do isolamento de
microrganismos que contribuem ou que prejudicam a formação dos grânulos e a
composição das SPE produzidas por consórcios de co-agregação.

3
 CAPÍTULO 1

Recent developments on aerobic sludge granulation technology

Abstract
Aerobic granules are large, dense biological aggregates that can be utilized in wastewater
treatment plants. The positive characteristics of this process such as high sedimentation
velocity, biomass retention, and resistance to toxic substances has attracted great interest
from researchers. Despite this intense research, the mechanisms responsible for aerobic
granulation and the effects of different operational and environmental factors are not well
understood. This review attempts to address recent developments in sludge aerobic
granulation based on the literature, focusing on factors that influence granule formation,
development, and stability. The possibilities of using aerobic granules in membrane
bioreactors (MBR), at high temperatures to remove nutrients and toxicity are also
presented. Finally, applications of granular aerobic sludge in full-scale treatment plants
are addressed.

Keywords: aerobic granular sludge, granule structure, granule formation, MBR with
granular sludge, full scale

1. Introduction
Granular sludge was first described in strict anaerobic systems in the late 1970s,
however the formation and application of aerobic granules was only observed in 1991
(ADAV et al., 2008; SARMA; TAY; CHU, 2016). Granulation is a gradual process that
transforms flocculent sludge into granular sludge by simultaneous densification and
selection of aggregates through settling (CAUDAN et al., 2014). The higher settling
velocity allows the reduction of secondary clarifiers and reduces by up to one-fifth, the
area required by conventional wastewater treatment plants (SARMA; TAY; CHU, 2016).

Anaerobic granulation exhibited several drawbacks that include the long

formation period required, the unsuitability for low organic load wastewater, and low
efficiency for the removal of nutrients (N and P) from wastewaters (ADAV et al., 2008).
In order to overcome these deficiencies in the anaerobic granulation process, research
efforts were devoted to aerobic granulation for wastewater treatment (QIN; LIU; TAY,
2004), making it a popular topic of discussion in the field of wastewater treatment
(ADAV et al. , 2008).

4
 The morphology of aerobic biogranules is completely different from any other
aggregate, including the flocs of conventional activated sludge. Biogranules are spherical
or rounded (MORAIS et al., 2016) with a regular, compact structure. They have an
excellent settling ability, and a strong resistance to high organic loads. Aerobic
biogranules are composed of a diverse microbial community that enables high metabolic
activity and greater tolerance to toxicity (ADAV et al., 2008; CAUDAN et al., 2014;
KOCATURK; ERGUDER, 2016; SAJJAD; Al. 2013). In addition, aerobic granulation
has been recommended for the treatment of high strength organic wastewaters containing
nitrogen, phosphorus, toxic substances, and xenobiotics (ADAV et al., 2008; CAUDAN
et al., 2014, CHEN et al., 2016, KOCATURK, ERGUDER, 2016).

Granulation is influenced by several parameters and operatinal conditions. Type

of seed sludge used, substrate composition, organic load rate, food to microorganism
ratio, feeding strategy, reactor design and hydrodynamics, settling time, exchange ratio,
presence of divalent cations, and aeration intensity are the most important among these
(ADAV et al., 2008; LIU, YU; WANG; TAY, 2005; SAJJAD KIM, 2015b).

The two major granulation selection pressures in a batch reactor are the settling
time and the volume exchange ratio. Granules can be selected according to their settling
velocity. Settling velocities lower than 5 m.h-1 and exchange ratios greater than 60%
increase the fraction of granules in the biological sludge (LIU, YU; WANG; TAY, 2005).
Granular sludge formation is encouraged by slow bacterial growth and the presence of
high shear forces in the bioreactor (CAUDAN et al., 2014).

Currently, most aerobic granulation research is still restricted to the treatment of

laboratory synthetic wastewaters. The results of these studies have many limitations for
practical applications. Thus, the benefits of treatment with granular aerobic sludge must
still be demonstrated with real and complex wastewaters (LONG et al., 2014).

Nevertheless, the quantity of research into wastewater treatment utilizing aerobic

granular sludge has grown rapidly around the world (AWANG; SHAABAN, 2016; WEI
et al., 2014). Granular aerobic sludge research has been extensively undertaken for the
treatment of various types of wastewaters, including effluents containing nitrogen,
phosphorus, and toxic substances. Due to these advantages, granular aerobic sludge may
be considered one of the most promising biological treatment technologies of the 21st
century (LI, KAI et al., 2015).

5
 This study presents a broad literature review regarding recent scientific
developments on aerobic sludge granulation. The aim of this review is to provide an
understanding of granule formation, its applications in nutrient removal systems, and in
membrane bioreactors under mesophilic and thermophilic conditions. Furthermore,
recent information on technological applications that use granular aerobic sludge on a full
scale are presented.

2. Formation of granular aerobic sludge

Granular aerobic sludge can be obtained under certain operating conditions.
Progress has been made in understanding how granulation occurs and how the process
can be accelerated, although granulation mechanisms are still not well understood
(ZHANG, QUANGUO; HU; LEE, 2016).

The process of aerobic granule formation is a crucial step for its applicability in
wastewater treatment plants given that a conventional activated sludge can take several
weeks to form biogranules (IVANOV et al., 2006; MALIK, A et al., 2003). Aerobic
granules can grow from different carbon sources and are formed under different organic
loads (LIU, YU et al., 2004).

Aerobic granules are widely investigated for the treatment of domestic and
industrial wastewater despite instability problems. The success of the application of this
process demands a better understanding of the composition of the wastewater and its
effects. An important parameter is the carbon / nitrogen (C / N) or chemical oxygen
demand / nitrogen (COD / N) ratio present in the effluent. The control of the COD / N
ratio can be carried out to exert microbial selection pressures to favor heterotrophic
bacteria (aerobic or anaerobic) or nitrifying species, contributing to granulation and the
efficiency of the system (WU et al., 2012).

The specific growth rate of aerobic granules is lower than that of flocculent sludge,
i.e., competition harms the formation and growth of aerobic granules and eventually
causes granular aerobic sludge eradication if the flocculent sludge is not effectively
drained from the bioreactor (LIU, YU; WANG; TAY, 2005).

The most significant factors that favor slow organism growth and granule
production in an aerobic reactor are the presence of feast and famine conditions, high
shear force, and short settling time. Thus, granular aerobic sludge has been produced
almost exclusively in sequential batch reactors (SBR), and may take from one week to

6
several months to develop mature granules with stable COD removal efficiency
(SARMA; TAY; CHU, 2016).

The SBR operating cycle consists of feeding, aeration, sedimentation, and effluent
removal. Increasing the cycle from 1.5 to 8 hours reduced the specific growth rate of the
biomass, while granules grown in cycles of 1.5 hours presented a larger size and those
cultivated in cycles of 4 hours were the most compact (LIU, YONG QIANG; TAY, 2007).

Fast-settling granules are dense spherical aggregates, while slowly settling

particles are small, low density, and irregular in shape, which allows the selection of
granules by the settling velocity (NI; YU, 2010). The short settling time causes the
washout of poorly settling suspended biomass, only retaining well settling granules
(ADAV; LEE; LAI, 2008).

The relationship between settling velocity (vsett) and the ratio of biomass of
aerobic granules to total biomass indicates that the fraction of aerobic granules in the
reactor increases with the increase in vsett. When vsett values are lower than 1 m.h-1, only
suspended bioflocs are cultivated and no aerobic granules are developed. As vsett increases
above 1 m.h-1, a blanket of aerobic granular sludge starts to form and prevails over the
suspended flocs at vsett values above 4 m.h-1 (LIU, YU; WANG; TAY, 2005). This
indicates that if the SBR is operated at values below the settling velocity of suspended
flocs (3 to 5 m.h-1), the flocculent sludge will not be effectively washed out of the reactor.

Improved granulation can be achieved by manipulating operating conditions

(LIU, YONG QIANG; TAY, 2015). Granules were formed in only 7 days using a 5-
minute settling time from the reactor start up rather than a gradual reduction of settling
time (QIN; LIU; TAY, 2004; QIN; TAY; LIU, 2004).

The increase in the organic load stimulates the secretion of extracellular polymeric
substances (SPE) and enhances aerobic granulation (LIU; LIU; TAY, 2004). The
application of a high hydraulic selection pressure such as a short cycle time (30 min), a
short settling time (1 min), and a high organic load (24 kg COD.m-3.d-1) resulted in the
formation of granules after 7 hours (ZHANG, XING et al., 2013). However, the granules
formed by this method only lasted 12 days in the reactor. The high organic loading rate
implies the formation of granules with low resistance and compromises the operation.
The long-term stability of the granules formed using the rapid formation strategy can be
obtained by reducing the organic loading rate after reaching the steady state (LIU, YONG
QIANG; TAY, 2015).

7
 The granulation mechanism comprises four steps, which are cell-to-cell contact,
micro-aggregate formation, excess production of EPS, and hydrodynamic compacting of
the granule matrix (Figure 1) (LIU, YONG QIANG; TAY, 2015). The use of granular
activated carbon (CAG) as a support medium increases the aggregation rate by favoring
the micro-aggregate formation step (ZHANG, QUANGUO; HU; LEE, 2016). The
presence of divalent and trivalent cations, such as Ca2+, Mg2+, Fe2+ and Fe3+ can bind to
negatively charged cells to form microbial nuclei (SOBECK; HIGGINS, 2002; WAN et
al., 2015).

Figure 1 –Mechanisms of formation of aerobic granules. Source: (SARMA; TAY; CHU, 2016).

The bacterial community present in the activated sludge influences the aerobic
granulation process. Hydrophilic bacteria have a lower tendency to attach to sludge flocs
than their hydrophobic counterparts do. Thus, the greater the number of hydrophobic
bacteria in the sludge the faster aerobic granulation occurs (ADAV et al., 2008). The role
of seeding sludge in aerobic granulation shows that the temperature at which the sludge
was produced influences the process. Granulation did not occur in some systems in which
sludge samples were collected at temperatures below 30 °C due to the absence of
Brevundimonas sp. (CHEN, LEE, 2015).
Biological reaction processes in aerobic granules are determined by oxygen
concentration gradients and by the types of substrate used. The substrate and dissolved
oxygen (DO) concentration profiles are a result of many factors, e.g., diffusion
8
coefficient, conversions rate, granule size, biomass spatial distribution and density (NI,
YU, 2010). All of these factors strongly influence each other and the effect of individual
factors therefore cannot be studied in isolation.
The aeration period of a SBR operation consists of two phases: a degradation
phase in which the substrate is depleted to a minimum, followed by an aerobic starvation
phase. The absence of carbonaceous organic matter contributes to increased
hydrophobicity, although it is not a prerequisite for granulation (ADAV et al., 2008).
Strong shear forces with higher aeration rates do not only compact aggregates into
granules, but also provide enough oxygen to suppress filamentous growth, contributing
to the long-term operational stability of a bioreactor (ADAV et al., 2008). Three SBR
with different aeration rates (1-3 L.min-1) fed with wastewater containing phenols were
compared. No granules formed at low aeration rates (1 L.min-1), while at high aeration
rates (3 L.min-1) stable and mature granules (1 - 1.5 mm) with a compact interior formed.
At an intermediate rate (2 L.min-1), large granules (3 - 3.5 mm) with overgrowth of
filaments were formed. An intermediate rate of aeration was not sufficient to meet oxygen
requirements or to inhibit filamentous growth, leading to bioreactor failure (ADAV; LEE;
LAI, 2007).
The substrate also influences the formation of granular aerobic sludge. The use of
propionate as a source of carbon delayed the granulation process compared to that with
acetate. However, the use of propionate yielded stronger granules (ZHANG,
QUANGUO, HU, LEE, 2016).
The increase in temperature contributes to granule formation. Reactors operated
at temperatures below 20 °C led to the formation of irregular shaped granules with
filamentous bulking, causing the loss of biomass and unstable operations. In addition,
denitrification capacity and nutrient removal rate of the granules at low temperatures were
reduced (ADAV et al., 2008). Temperatures above 40 °C hamper granule formation
(EBRAHIMI et al., 2010; LÓPEZ-PALAU et al., 2013).

2.1. Extracellular polymeric substances

Extracellular polymeric substances (EPS) are important for microbial aggregate
formation, since bacteria are dispersed in an EPS matrix. These substances have a
structural function and serve as a protective barrier and affect porosity, density,
hydrophobicity, mechanical stability, and other properties of biofilms (LIU, YONG
QIANG; TAY, 2007).

9
 Bacterial EPS are associated with the formation of aggregates and cell-to-cell
bonds (REN; XIE; XING, 2009). EPS expelled by microorganisms during growth are
crucial for granule formation and maintenance. Proteins and polysaccharides are the main
components of EPS containing large amounts of functional groups such as hydroxyl and
carboxyl (CAUDAN et al., 2014; KOCATURK; ERGUDER, 2016; SARMA; TAY;
CHU, 2016). These functional groups have a high binding capacity and are closely
associated with the stability of microbial aggregates (LIU, YONGJUN et al., 2014;
SAJJAD; KIM, 2015b).

The main characteristics of aerobic biogranules depend on their EPS composition.

Hydrophobic proteins are the major components of EPS and therefore an increase in EPS
production results in increased hydrophobicity, density, and settleability of the granules
(CORSINO, SANTO FABIO et al., 2016). EPS contribute to cell adhesion and formation
of a microbial matrix of the granules, improving their stability and structural integrity
(LIU, YONGJUN et al., 2014). Although EPS are essential for aerobic granulation,
excessive levels reduce granule stability (CORSINO, SANTO FABIO et al., 2016).
Excess EPS production transforms aerobic granules into a gelatinous structure,
characterized by a dense, viscous surface layer that limits oxygen and nutrient transfer,
which in turn reduces bacterial metabolic activity. The reduction of the porosity of the
granules also limits the transport of nutrients to the inner layers of the granules, causing
bacterial death and rupturing of the granules over the long term (CORSINO, SANTO
FABIO et al., 2016).

The composition of EPS in terms of its main components, proteins and

carbohydrates, influences the characteristics of the granules formed. A low
polysaccharide/protein (PS/PN) ratio results in the deterioration of strength and
settleability and disintegration of the granules. Ratios less than 0.6 may cause structural
instability and granular disintegration (KOCATURK; ERGUDER, 2016).

The production and consumption of EPS increases according to the cycle time of
of the SBR. Bacteria use these products as a source of carbon and energy when subjected
to long periods without feeding, decreasing the amount of EPS and proteins as the cycle
time increases (CORSINO, SANTO FABIO et al., 2016).

Proteins and polysaccharides are the major constituents of EPS and can account
for 75 to 90% of EPS mass, while humic substances, uronic acids, and nucleic acids are
present in smaller amounts (BASUVARAJ; FEIN; LISS, 2015 YAN, LILONG et al.,
2015).
10
 Proteins are an important component for changes in the structure of the flocs,
contributing to the increase of settling velocity and favoring the formation of aerobic
granules (BASUVARAJ; FEIN; LISS, 2015; SAJJAD; KIM; KIM, 2016). The proteins
and their amino acid composition contribute to the hydrophobic character of the flocs.
The high content of negatively charged amino acids allows a greater amount of
electrostatic bonds of multivalent cations with proteins than with polysaccharides,
increasing the stabilization of the aggregate structure (BASUVARAJ; FEISS, LISS,
2015; CAUDAN et al., 2014). Protein functions include the aggregation of bacterial cells
and the formation of an active gel-like matrix that maintains cell cohesion
(BASUVARAJ; FEIN; LISS, 2015).

Polysaccharides are related to the formation of a gel-like layer more favorable to

the formation of biofilms than granules (BASUVARAJ; FEIN; LISS, 2015).
Polysaccharides are hydrophilic polymers that absorb and contribute to a high retention
of water or biological fluids, resulting in poor settling and poor dewatering of the sludge
(BASUVARAJ; FEIN; LISS, 2015; SAJJAD; KIM, 2015b). For the granules, a high or
excessive polysaccharide content is detrimental to the settling and dewatering properties
due to a higher water content associated with polysaccharide-rich EPS (BASUVARAJ;
FEIN; LISS, 2015).

The amount of EPS, especially proteins, increases significantly during the

transition from flocculent to granular sludge (CAUDAN et al., 2014). The use of
flocculent sludge with higher polysaccharide content (ratio PN/PS = 0.5) showed a
change in EPS composition during granule formation. The granular aerobic sludge
presented a higher protein content (ratio PN/PS = 1.6) in its total EPS fraction
(BASUVARAJ; FEIN; LISS, 2015). The proteins were located predominantly in the core
region of the granules where the cells were tightly packed (BASUVARAJ; FEIN; LISS,
2015; CAUDAN et al., 2014).

The PN content and PN/PS ratio show a positive correlation with the particle size
of the granular sludge. The protein content gradually increases during the formation of
the granular sludge while the polysaccharide content remains practically constant (YAN,
LILONG et al., 2015).

The EPS content may also vary depending on the presence of toxic substances.
The addition of 20 mg.L-1 of 2,6-dichlorophenol in a synthetic effluent increased PN
concentration by about 30 fold while the PS concentration remained more or less constant
(LI, KAI et al., 2016).
11
 2.2. Divalent cations addition
The presence of divalent cations improves the physical (particle size, settleability,
filtrability etc.) and chemical (EPS) characteristics of the granules. Several authors
observed a shorter time for granule formation in systems with added calcium (LIU,
YONGJUN et al., 2014, SAJJAD, KIM, 2015b, SOBECK; HIGGINS, 2002). The
positive charge of divalent cations neutralizes the negative charge of the microbial
biomass surface and EPS molecules thereby enhancing granulation (SAJJAD; KIM,
2015a, b; SOBECK; HIGGINS, 2002). These cations can stimulate granulation by
neutralizing the negative charges present on bacterial surfaces, implying strong van der
Waals attractive forces, or functioning as cationic bridges between bacteria given that
most microorganisms are negatively charged at the typical pH of bioreactors (LIU, YU et
al., 2004).

Added calcium improves settleability, and increases the amount of EPS produced
by the granules. Magnesium, on the other hand, also shows a positive correlation with
granule formation, but the effects of Mg2+ are lower than those of Ca2+. In the monitoring
of parallel systems, the addition of calcium caused a greater increase in particle size and
reduction of the sludge volume index (SVI) when compared to systems that received
added magnesium. This was due to the increase of polysaccharides synthesis in EPS in
the presence of Ca2+ (SAJJAD; KIM, 2015b). Added calcium increases the strength of
the granules and the addition of magnesium increases granule microbial diversity
(CAUDAN et al., 2014). The addition of Mg2+ favors nucleation of the granules and Ca2+
addition favors growth and maintenance of a more rigid structure. The simultaneous
addition of Mg2+ during nucleation and Ca2+ in the granule-growth stage accelerates the
granulation process more than the addition of either cation alone (CAO et al., 2014).

Metal ions accelerate granulation by forming an initial nuclei for bacterial

attachment (LIU, YONG QIANG; TAY, 2015). Denitrification is indicated as beneficial
to the process due to the formation of CaCO3 (ZHANG, QUANGUO; HU; LEE, 2016).
Calcium carbonate accumulates inside the granule forming a rigid structure, thereby
increasing granule strength (CHEN et al., 2016).

Cellular polysaccharides are adhesives that facilitate cell cohesion and adhesion,
causing an increase in the granule diameter (SAJJAD; KIM, 2015b). The cohesion of the
granules depends on the establishment of calcium bridges between anionic proteins that
increase the size and compactness of the aggregates. Calcium bridges are more specific
12
for anionic proteins than for anionic polysaccharides, and are more frequent in the
presence of divalent cations than monovalent cations (CAUDAN et al., 2014; SARMA;
TAY; CHU, 2016).

Analysis of the interactions between the divalent cations and the EPS constituents
revealed that calcium binds preferentially to hydroxyl groups (OH) bound to the
polysaccharide carbons than to nitrogen (N) of amide group of proteins. Magnesium, on
the other hand, exhibits greater interaction with N of amide groups of proteins than with
polysaccharides. This behavior can be explained by (i) the specific position of the
functional groups present in protein and polysaccharide molecules and (ii) the larger size
of Ca2+ in relation to Mg2+ (SAJJAD; KIM, 2015b).

Divalent cations retain small particles and increase resistance of the three-
dimensional matrix of the exopolymers, causing an increase in granule size and
dewaterability. Added calcium leads to an increase in the production of polysaccharides
by the microorganisms in the granules, while the addition of magnesium has a greater
effect on protein production. Proteins are the hydrophobic constituents of EPS that
facilitate the dewatering of sludge. It is observed that the use of different divalent metals
changes the composition of EPS, although their total amounts are almost similar. While
Ca2+ mainly affects the size and settleability of the granules, Mg2+ has a greater effect on
dewatering (SAJJAD; KIM, 2015b).

Protein amide groups are surrounded by carbon atoms, which are attached to high
molecular weight alkyl and aryl groups. In addition, these groups are in opposite positions
(trans position) to the amide group. The larger size of the Ca2+ ions makes it difficult to
bind to amide N or O of the amide groups due to steric hindrance, whereas it is easier for
calcium to interact with the readily available polysaccharides hydroxyl groups.
Magnesium, because it is much smaller than calcium, encounters no steric hindrance in
binding with the N of protein amide groups and fits well within the structure of the
polymer, implying a higher protein retention in the presence of Mg2+ (SAJJAD; KIM,
2015b).

Surface charge neutralization and aggregation enhancement can also be achieved

by the addition of trivalent cations, such as aluminum. The effect of poly aluminum
chloride (PAC) on the chemical properties of the granular sludge showed an increase in
hydrophobicity and a reduction in the zeta potential of granular sludge, by lowering
electronegativity of the suspended solids surface charge and increased EPS production.

13
Microorganisms secrete EPS to protect themselves against osmotic pressure in the
presence of cations (LIU, YONGJUN et al., 2014).

PAC addition increases the re-formation of aerobic granular sludge that

disintegrates spontaneously after a long period of batch reactor operation. Granules are
formed in less time and are stronger than granules formed without the addition of PAC
(LIU, YONGJUN et al., 2014).

The efficiency of divalent cation addition is influenced by the presence of

chelating agents, which may impair the granulation process. Chelating agents dissolve
calcium precipitates and destabilize the granules. Addition of phosphate ions (PO43-)
reduces formation of EPS ionic bridges (CORSINO, SANTO FABIO et al., 2016;
SARMA; TAY; CHU, 2016). Phosphate solubilization by acids present in the wastewater
or by microorganisms during biological removal of phosphorus causes aerobic granule
destabilization (SARMA; TAY; CHU, 2016).

Addition of 1 to 5 mg.L-1 Cu2+ does not affect structural integrity, but makes
aerobic granules less compact. However, granules disintegrate with the addition of 10
mg.L-1 of Cu2+due to the reaction of the metal ions with the -NH2, -OH, -COOH and C-
N functional groups. The increase in Cu2+ concentration from 0.0 to 10.0 mg.L-1 leads to
the decrease in weight percentages of essential elements Fe, Ca, Na, and K in the granules
while that with copper increases correspondingly. Cu2+ is exchanged with the essential
metals and chelated by the nitrogen functional groups of the protein, reducing the
structural stability of the sludge (ZHENG et al., 2013).

The presence of chelating agents may also improve granular aerobic sludge
formation. Aerobic granulation was greater in three SBR that were fed with added
nitrilotriacetic acid (NTA) compared to the reactor fed with acetate in the absence of
NTA. NTA contributed to an increase in selective pressure for the enrichment of the
granules. Acetate is easily degradable and the presence of NTA reduced the growth rate
of the granules and allowed the enrichment of slow growing microorganisms in the
reactors fed with NTA. The chelating agent may have exerted a strong detachment force
on the granule surface. This effect resembles that of the shear force and contributes to the
formation of denser granules (NANCHARAIAH et al., 2008).

Chelating agents reduce the formation of biofilms at high concentrations. The

addition of 50 mM of ethylenediamine tetraacetic acid (EDTA) reduced the number of
biofilm-associated cells by more than 99% (BANIN; BRADY; GREENBERG, 2006).

14
However, it likely increases biogranulation at lower concentrations. Granules formed in
the presence of 0.26 mM, 0.52 mM and 1.05 mM of nitrilotriacetic acid (NTA) were
smoother, denser, more compact, and showed better settling characteristics than those
formed in its absence (NANCHARAIAH et al., 2008). The role of chelating agents in the
formation of granular aerobic sludge requires further investigation.

2.3. Microbial coaggregation

The formation of granules can be obtained by selecting microbial cultures. When
microorganisms are inoculated into the sludge, they can remain in the reactor for a long
period of time, helping to form aggregates and maintaining the degradative capacity of
these organisms (IVANOV et al., 2006).

The addition of a pure culture with high self-aggregative capacity to an activated

sludge resulted in the formation in only eight days of aerobic granules with a mean
diameter of 446 μm (IVANOV et al., 2006). A rapid reactor start-up for solvent recovery
was achieved by the inoculation of a mature granular sludge and the implementation of
an aerobic biological selector (LONG et al. 2016).

Coaggregation offers several advantages for bacteria. It avoids cell washout and
protozoan predation, resistance to toxins, better transfer of chemical signals; exchange of
genetic information and better provisions for carbon and energy sources (MALIK, A et
al., 2003; MALIK, ANUSHREE, KAKII, 2008; NAGAOKA et al., 2008).
Bioaugmentation with specific pure cultures accelerates aerobic granulation, although it
is a complex and costly process for practical application (LIU, YONG QIANG; TAY,
2015).

Physical interactions between aggregated bacteria facilitate metabolic

interactions, such as oxygen protection, cell-cell communication, and genetic exchange
between cells. Aggregation usually depends on highly specific lectin-carbohydrate
interactions occurring between the aggregating partners and can be defined as
interspecific (coaggregation) or intraspecific (self-aggregation) (LEDDER et al., 2008;
RICKARD; MCBAIN et al., 2003; RICKARD; GILBERT; HANDLEY, 2004).

Coaggregation interactions contribute to the development of biofilms through two

routes. The first route is via single cells in suspension specifically recognizing and
adhering to genetically distinct cells in the developing biofilm. The second route is by the
prior coaggregation in suspension of secondary colonizers followed by the subsequent

15
adhesion of this coaggregate to the developing biofilm (RICKARD & GILBERT et al.,
2003). However, the mechanisms that control interactions in biofilms containing several
species has yet to be determined (SIMÕES; SIMÕES; VIEIRA, 2007).

From the standpoint of mass transfer and substrate utilization, it is more

advantageous for the bacteria to remain dispersed than to form aggregates. However,
physico-chemical properties of the cell surface have great effects on the formation of
biofilms. The hydrophobicity induced by conditions of the medium can function as a
factor that triggers aerobic granulation. As the cell becomes more hydrophobic, cell
adhesion is observed, that is, cell surface hydrophobicity should influence the ability of
cells to aggregate. On the other hand, a change in the chemical composition of the
wastewater can cause the loss of the established granular sludge (LIU, YU et al., 2004).

Cell hydrophobicity can be induced by environmental conditions and enable cell-

cell aggregation, which is crucial for the formation of biological granules (EKMEKCI;
ASLIM; OZTURK, 2009; MALIK, ANUSHREE; KAKII, 2008; PHUONG; KAKII;
NIKATA , 2009). Thermodynamically, microbial aggregation is controlled by the
reduction of free energy. The increase in the hydrophobicity of cell surfaces reduces
Gibbs energy from the surface, which promotes cell-to-cell interaction and subsequently
leads to aggregate formation (QIN; TAY; LIU, 2004).

The association among bacteria is highly specific and primary colonizers may
aggregate with each other, but do not readily aggregate with secondary colonizers.
However, some species of secondary colonizers may aggregate with both primary and
secondary colonizers, acting as bridge organisms (BUSWELL et al., 1997; MALIK et al.,
2003; NAGAOKA et al., 2008; RICKARD GILBERT et al., 2003). In the absence of this
type of organism other secondary colonizers cannot be part of the biofilm (RICKARD;
GILBERT et al., 2003).

2.4. Important microorganisms in granulation processes

Several microorganisms that act in the formation of aerobic granules have been
reported in the literature. Factors such as temperature, nutrients, and salinity are crucial
for the selection of microorganisms favorable to granulation.

The bacterium Blastomonas natatoria 2.1 coaggregated specifically with all 18

other bacteria isolated from an original freshwater community. B. natatoria 2.1 might
have a role as a bridging organism in the development of the biofilm community

16
mediating adhesion between primary and secondary colonizers (RICKARD; GILBERT
et al., 2003).

The sampling of biological sludge at different temperatures throughout the year

in the same wastewater treatment plant showed different behavior in relation to the
formation of granules. Sludge collected at lower temperatures did not form granules. A
comparison of the sludge over the year showed that no granules were formed in the
samples lacking Brevundimonas sp., an effective producer of EPS that has an optimal
growth temperature of 30 ℃ (CHEN, LEE, 2015). This indicated the importance of this
EPS producer in granulation.

The microbial composition of granules varied as a function of temperature in a

biotreatment aimed at the removal of phosphorus. Filamentous bacteria Leptothrix spp.
were more abundant at 20 °C than at higher temperatures (30 °C and 35 °C). Removal of
phosphorus was insignificant and there was a greater abundance of
Gammaproteobacteria at 30 and 35 °C. Removal of phosphorus at 20 °C was related to
the presence of Rhodocyclaceae. The presence of Sphingomonadaceae at all temperatures
indicated that this family played an important role in the maintenance and stability of
aerobic granules (EBRAHIMI et al., 2010).

The evolution of the bacterial population during formation of granular sludge for
removal of phosphorus and denitrification indicates an increase in microbial diversity and
richness during the granulation process, followed by a stabilization of these parameters
upon the presence of granules. Betaproteobacteria, Gamaproteobacteria and
Deltaproteobacteria are indicated as groups that contribute to the increase in mechanical
resistance and formation of granular aerobic sludge. Chloroflexi excel contributes to
carbohydrate biodegradation, nitrification, and denitrification and Actinobacteria are
important for COD removal (HE et al., 2016). Proteobacteria and Bacteroidetes phyla
are the predominant bacteria and have the best adaptation to changes in growth
environment (SONG et al., 2009; ZHAO et al., 2015).

The study of partial nitrification in wastewater with high concentrations of

ammonia indicates that Nitrosomonas europaea and Nitrosomonas sp. are the bacteria
responsible for ammonia oxidation, whereas Nitrobacter sp. and Nitrospira cf.
Moscoviensis oxidize nitrite. Reduction of treatment temperature and carbon source
concentration decreases the nitrification performance and there is a predominance of
ammonia-oxidizing bacteria (AOB) in the microbial community of the granules. At the
same time, increasing the temperature favors nitrite-oxidizing bacteria (NOB), allowing
17
them to compete with the AOB in the granules (WAN et al., 2014). Nitrosomonas were
the major ammonia-oxidizing bacteria in the sludge used as inoculum, but after the
formation of the granules, Nitrosomonas and Nitrosospira were present in equal amounts
(WINKLER et al., 2013). The variation of reactor operating temperature indicated that
ammonia was oxidized by Accumulibacter sp., Thauera sp., Tetrasphaera-PAO and
Azoarcus-Thauera, regardless of temperature (20 and 25 °C) favored the occurrence of
denitrifying bacteria such as Comamonadaceae, Curvibacter sp., Azoarcus cluster,
Rhodobacter sp., Roseobacter sp. and Acidovorax spp. (MIECZKOWSKI et al., 2016).

The salinity effect on the microbial community present in aerobic granules was
evaluated and the main classes identified in low salinities were Alphaproteobacteria,
Cytophagia and Betaproteobacteria, while the classes Alphaproteobacteria,
Flavobacteria and Actinobacteria prevailed in high salinity. The presence of
Alphaproteobacteria under different conditions is related to the metabolic ability to adapt
and survive under stress conditions. Bacterial strains of EPS producing-bacteria were
found in both aerobic granules at low and high salinity conditions, indicating that EPS
production is important for aerobic granulation at high salinity (RAMOS; SUÁREZ-
OJEDA; CARRERA, 2015).

In a study of the microbiological composition of the granules, it was found that

mature granules have an anaerobic spherical core with Rhodocyclaceae covered by an
outer spherical shell with both aerobic and anaerobic strains (LV et al., 2014). The
development of granules indicated an increase in the abundance of the anaerobic species
Flavobacteriaceae, Xanthomo nadaceae, Rhodobacteraceae and Microbacteriaceae, due
to the formation of an anaerobic core (LV et al., 2014). Strains of Sphingomonas sp.,
Paracoccus sp., Sinorhizobium americanum, and Flavobacterium sp. present in the core
promoted the production of EPS and improved the formation of granules (WAN et al.,
2015).

The presence of filamentous bacteria in aerobic granules at low levels improves

stability and mineral capture by the granules, contributing, for example, to enhancing
phosphorus removal (LI, YONGMEI et al., 2014). However, the excess of these
microorganisms may impair treatment with granular aerobic sludge. The predominance
of filamentous bacteria in aerobic granules modifies their structure, making them loose,
fluffy, and irregular and leads to the disintegration of granules (CORSINO, S. F. et al.,
2016). The outgrowth of filamentous bacteria on the granule surface reduces the settling
velocity of the granules and results in sludge wash out (LIU, HONGBO et al., 2014). The
18
increase in temperature in the bioreactor from 30 to 50 °C causes the elimination of
filamentous bacteria in aerobic granules (AB HALIM et al., 2015).

2.5. Granule stability

The loss of structural stability and bioactivity after long-term operations are the
main challenges to the application of aerobic granular processes (ZHANG, QUANGUO;
HU; LEE, 2016). The mechanical strength of granules depends on their growth conditions
and the substrate (CAUDAN et al., 2014). The formation of granules is stimulated by
slow-growing bacteria and the presence of high shear forces (CAUDAN et al., 2014; LIU,
YONG QIANG; TAY, 2015). Rapid-growth bacteria lead to instability of the aerobic
granules (LIU, YONG QIANG; TAY, 2015). The presence of a recalcitrant substrate
leads to a lower growth rate of heterotrophic bacteria, with the selection of slow-growing
bacteria prevailing, increasing the long-term stability of the aerobic granules (CAUDAN
et al., 2014; LIU, YONG QIANG; TAY, 2015).

One of the most serious barriers to practical applications of aerobic granules is the
loss of granule stability over long-term operation, caused by granule break-up or
filamentous bacteria overgrowth. In the first case, the granules deteriorate into small
pieces and are washed out. In the second case, the growth of filamentous bacteria
produces low density granules that are easily washed out (ADAV et al., 2008).

Filamentous bacteria overgrowth, reduction of nutrient transfer into the granules,

the formation of anaerobic zones in the aggregates, and EPS properties may result in
granule disruption (KOCATURK; ERGUDER, 2016). Maintenance of alkaline
conditions avoids excessive growth of filamentous bacteria that cause granule
disintegration and biomass washout (ZHANG, QUANGUO; HU; LEE, 2016). The
increase in the duration of the feeding phase is related to the loss of stability of aerobic
granules due to the excessive growth of filamentous bacteria (CORSINO, SANTO
FABIO et al., 2016).

Aerobic granules can also disintegrate under high organic load. An increase in
organic loading rate can increase granule size and enhance the growth of anaerobic cores
inside the granules. The dead cells within the granules cause the collapse of the granular
structure (ZHANG, QUANGUO; HU; LEE, 2016). In systems with chemical oxygen
demands (COD) greater than 3000 mg.L-1, the isolates lost their capacity for self-
aggregation and the production of proteins and polysaccharides. The reduced protein

19
quantities excreted by the isolates were associated with the low integrity of the granules
under high organic loadings (ADAV; LEE; LAI, 2010).

Aerobic granule stability may be associated with one or more characteristics of

the granules, such as morphology, settleability, density, strength, surface hydrophobicity,
specific oxygen uptake, microbiological structure and diversity and EPS content (LEE et
al., 2010). Granule rupture is caused by mineralization of their core, as well as the
clogging of their pores, caused by excess EPS that limits the flow of nutrients and oxygen
from the bulk into the inner layers (CORSINO, SANTO FABIO et al., 2016).

A strategy to achieve granule stability is the selection of flocculating

microorganisms through a short settling time and a high height/diameter ratio of the
cylindrical bioreactor. The application of intermittent feeding or the occurrence of a low
feed availability period in the bioreactor cycle increases the hydrophobicity of the cell
and increases granulation. A high aeration rate provides sufficient dissolved oxygen and
benefits cell aggregation. Low pH and temperatures should be avoided to prevent fungal
growth. The presence of divalent cations helps cell granulation. Also, high shear forces
contributes to the formation of a compact core (LEE et al., 2010). Microbial selection
pressure is not a prerequisite for granulation. The microbial community is similar to that
of the sludge used as an inoculum, and the bioflocs detach, collide, and aggregate
randomly and continuously with granules (ZHANG, QUANGUO; HU; LEE, 2016).

Among the factors involved in the formation and disintegration of granules,

granule size is considered essential (LIU, YONGJUN et al., 2014), because granule size
plays an important role in limiting mass transport and diffusion. The increase in size and
age of the granule reduce the porosity of these structures (YONGJUN et al., 2014).
Aerobic granules become unstable after they reach a certain dimension. Indeed, once this
occurs, these granules breakdown and most of them are washed out, while others
aggregate again forming new granules. Over the long term, granulation is a dynamic
process in which granulation and degranulation continuously take place (CORSINO,
SANTO FABIO et al., 2016).

EPS excreted by micoorganisms during cell growth and lysis play an important
role in granule formation and maintenance (LIU, YONGJUN et al., 2014). Due to the
compact structure and large size, few substrates can penetrate into the interior of the
aerobic granule, causing EPS located at the core of the granule to be used as energy source
and the core biomass undergoes microbial decay and lysis (LEE et al., 2010). These

20
changes may eventually cause the structure to weaken and result in the disintegration of
the aerobic granules by hydraulic shear forces.

2.6. Nutrient removal

One of the major problems related to granular aerobic sludge technology is to
obtain uniform conditions for nutrient removal. The bacteria responsible for the removal
of nitrogen are slow-growing microorganisms and the efficiency of nitrogen removal
depends on granule maturation and the adaptation of the autotrophic bacteria, reaching
efficiencies above 90% (CORSINO, SANTO FABIO et al., 2016)

Nitrogen removal efficiency depends on the availability of a carbon source for

denitrification (CORSINO, SANTO FABIO et al., 2016). The dense spherical structure
of the granules causes a limitation of oxygen transfer, resulting in an oxygen gradient in
the sludge granules and allowing denitrification (LI, YONGMEI et al., 2014). The
increase in the famine phase in an SBR can reduce the availability of carbon, creating a
very slow denitrification rate (CORSINO, SANTO FABIO et al., 2016).

Aerobic granulation can be obtained with COD/N ratios between 1 and 20

(KOCATURK; ERGUDER, 2016). The amount and activity of heterotrophs increases
with respect to nitrifiers at high COD/N values, for example equal to 20. Nitrifiers are
favored at lower COD/N ratios such as lower than 3.3 (KOCATURK, ERGUDER, 2016).
However, COD/N ratios lower than 2 may negatively affect the stability of the granules,
resulting in granule disruption at a COD/N equal to 1. The disintegration is due to the
degradation of the EPS matrix and microbial community change (LUO et al., 2014).

Bioreactors operating with a COD/N ratio higher than 10 are recommended for a
greater COD removal and with a ratio lower than 5 when further nitrogen removal is
desired. COD/N ratios ranging from 7.5 to 30 result in high COD removal efficiency
(92%) and reduced nitrogen removal (33%) due to a greater growth of heterotrophs
resulting in large, white, fluffy granules. Under these conditions, maintenance of high
treatment efficiency and granular stability is difficult due to significant heterotroph
growth rates. COD/N ratios between 2 and 5 result in a complete nitrification and in lower
COD removal (60%). The granules formed under these conditions are small, dense,
orange granules enriched in nitrifiers with slow-growing but stable characteristics. The
optimum value of the COD/N ratio in terms of removal of COD (75-79%) and nitrogen
(> 90%) is equal to 7.5 (KOCATURK, ERGUDER, 2016).

21
 Aerobic granules were used for the treatment of high-strength ammonium
wastewaters and it was found that granular adsorption and nitrification followed by
denitrification corresponded to 9% and 76%, respectively, of the total nitrogen removal
(YU et al., 2014). Application of granules in nitrification and denitrification processes
has been used in order to save energy and substrate (ZHANG, QUANGUO; HU; LEE,
2016).

Granules can be applied for simultaneous removal of nitrogen and phosphorus

(WEI et al., 2014). Aerobic granules grown at 50 ℃ had a mean diameter of 3.36 mm and
COD, ammonia and phosphate removal efficiency of 98.17%, 94.45%, and 72.46%,
respectively (AB HALIM et al. 2015). Phosphorus accumulated in the aerobic granules
due to chemical precipitation in the presence of the filamentous bacteria (genus Thiotrix)
(LI, YONGMEI et al., 2014; ZHANG, QUANGUO; HU; LEE, 2016). Older sludge age
of the granular system and the growth of filamentous bacteria increases the capture and
involvement of minerals in the granules (LI, YONGMEI et al., 2014).

Use of aerobic granular sludge for the treatment of wastewater with high
concentrations of ammonia and phosphorus presents many problems. The low growth rate
of nitrifying bacteria increases the time required to form granules and ammonia can
prevent the formation of granular sludge by inhibiting microorganism metabolism (WEI
et al., 2014). Rapid formation of granules is desirable, however it is important to maintain
a favorable condition for the growth of nitrifying bacteria to obtain a high nutrient
removal capacity (LOCHMATTER; HOLLIGER, 2014). In addition, the reduction of
nitrate to nitrogen gas during the anoxic phase in conventional biological nutrient removal
systems may inhibit phosphate release (WEI et al., 2014).

Phosphate removal in systems with granular aerobic sludge may occur chemically
or biologically. Biological phosphate removal is promoted by phosphate accumulating
organisms (PAO) and chemical removal can occur by dosing metal salts or by biologically
induced phosphate precipitation. Phosphate precipitation products such as struvite and
hydroxyapatite are especially interesting because they are common products used in
agriculture (STUBBÉ, 2016).

Phosphate accumulating microorganisms are important for the removal of

phosphorus, but precipitation is the main mechanism acting on systems with aerobic
granules. Increased pH during treatment and longer sludge retention time (SRT)
contribute to the accumulation of phosphorus in the aerobic granular sludge (LI,
YONGMEI et al., 2014). Accumulation of phosphorus in the granules was 150.7 ± 28.5
22
mg.gSS-1 of P (milligram of phosphorus per gram of suspended solids) in a reactor with
a high phosphorus concentration in the influent (35 mg.L-1 of P). In addition, the addition
of calcium (40 mg.L-1 Ca) increased the phosphate precipitation, augmenting the
phosphorus content in the sludge from 2.5% to 10% in 25 days (LI, YONGMEI et al.,
2014).

2.7. Biodegradation, bioaccumulation and biosorption

The removal mechanisms of toxic and harmful organic pollutants involve
adsorption, absorption, concentration, biotransformation, or microbiological degradation
(YAN, QING et al., 2016). The most promising feature of aerobic granules in treating
contaminants of emerging concern is the ability to treat pollutants simultaneously through
three environmentally friendly methods that may vary depending on the type of pollutant:
biodegradation, bioaccumulation, and biosorption. Granules are capable of providing the
advantages of aerobic and anaerobic biodegradation since they are composed of both
aerobic and anoxic/anaerobic zones (SARMA; TAY; CHU, 2016).

Microorganisms in aerobic granules may share biodegradation pathways similar

to any other mixed microbial cultures, where a degradation product generated by one
organism may be used by others to achieve complete degradation. Activated sludge
systems, on the other hand, do not completely degrade the contaminants of emerging
concern that are often found in effluents such as pharmaceutical drug residues and
endocrine disrupting substances (SARMA; TAY; CHU, 2016).

EPS is generally the first microbial cell barrier that interacts with toxic substances
and is closely related to the stability of aerobic granules (LI, KAI et al., 2015).
Comparison of the responses of nitrifying granules (NG) and conventional granules (CG)
to tetracycline (TC) indicated that more EPS were produced with higher protein content
than polysaccharides to protect against TC stress (SHI et al. 2013). Many organic
pollutants, such as benzene and phenanthrene dyes, can be absorbed by hydrophobic EPS.
Besides, EPS also contains active enzymes that may contribute to the removal of organic
contaminants through biological transformation (LI, KAI et al., 2015).

Persistent organic pollutants with high octanol-water partition coefficients (KOW)

tend to adsorb on the surfaces of the granules and can subsequently be removed. Aerobic
granulation has been considered a promising technology for the removal of heavy metals,
such as nickel, cadmium, copper, and zinc (DENG; SU, 2016; SARMA; TAY; CHU,
2016).
23
 Aerobic granules were used to treat wastewater containing phenol, o-cresol and
p-nitrophenol under high salinity conditions and the biogranules reached the complete
biodegradation of aromatic compounds (RAMOS; SUÁREZ-OJEDA; CARRERA,
2015). Treatment of organic pollution by pharmaceuticals and personal care products
indicated that biogranules promoted the growth of Proteobacteria, Bacteroidetes, Beta
Proteobacteria and Zooglea to remove some but not all of the PPCP tested (ZHAO et al.,
2015).

The residence time and concentration of RP4 (antibiotic resistance plasmid) in the
sludge decreases as the size of the aggregates increases. Smaller aggregates are more
vulnerable to changes and alien species, while larger aggregates with a diameter greater
than 0.9 mm maintain a relatively stable microbial composition and are immune to the
hazards of newly introduced bacteria. The granules have a compact formation and a high
density of microorganisms, and are considered an independent microbial ecological
system, which hampers invasion of the RP4 plasmid. Therefore, the use of the granular
sludge system can reduce the transmissions of ARG and reduce potential ecological
threats, thus improving the ecological safety of wastewater treatment processes (ZOU et
al., 2015).

The addition of 40 mg.L-1 of Bisphenol A (BPA), a highly toxic yet important

industrial chemical, to synthetic wastewater caused an increase in the amount of EPS,
especially proteins, produced by aerobic granular sludge (LI, KAI et al., 2015). A system
with aerobic granules showed an efficiency equal to 91.8% while a conventional activated
sludge removed 71.3% of the BPA present in the municipal wastewater from Bari, Italy.
These results may be explained by the very old sludge age (about 160 d) of the aerobic
granule system which offers a greater chance of removal of endocrine disruptors
(BALEST, LYDIA et al., 2008). The relatively high stability of these compounds in
municipal effluents is mainly due to the insufficient retention time of the sludge from
conventional biological systems (BALEST, L et al., 2008).

3. Membrane bioreactor with granular sludge

Since granules are dense aggregates larger than flocs they were found to be a
possible alternative to minimize the fouling problem in an MBR. It is possible to maintain
higher flow with granular sludge than flocculent sludge, due to the reduction in membrane
fouling, one of the main challenges for operation of MBR, causing a flux reduction and
increasing the costs of cleaning and regeneration of the membrane modules (LI, WEN
24
WEI et al., 2012; LI, XIUFEN et al., 2005; MORAIS; SILVA; BORGES, 2016; TU et
al., 2010; WANG et al., 2013).

Many factors affect membrane fouling, including sludge particles and structures
that can deposit and accumulate on the membrane surface to form a cake layer and large
soluble molecules plugging and narrowing the pores of the membrane. Aerobic granules
may contribute to delaying membrane fouling as they do not easily adhere to membrane
pores due to their large particle size (WANG et al., 2013).

Different fouling mechanisms, such as adsorption of macromolecules, pore

blocking or biofilm formation, can be established on the surface of the membranes.
Physiological characteristics of the sludge, such as the concentration of solids and the
presence of EPS, change according to the operating conditions of the bioreactor. Thus,
fouling processes in membrane bioreactors are unpredictable and difficult to control (LE
CLECH et al., 2003).

Mechanisms of membrane fouling in MBR differ according to the type of sludge.

Resistance due to the gel layer is the main factor for membrane fouling in systems
operated with flocculent sludge, while pore blocking is mainly responsible for the fouling
caused by granular aerobic sludge (WANG et al., 2013). The smaller particles in the
flocculent sludge accumulate rapidly on the surface of the membranes to produce a cake
under high flux due to the transmembrane pressure and high compressibility of the
biological flocs (WANG et al., 2013).

The size of the granular sludge particles is generally larger than the pore size of
the microfiltration membranes. Particles larger than 100 μm corresponded to 20% and
90% of the flocculent and granular sludge, respectively (WANG et al., 2013). The
presence of EPS contributes to the production of granules larger than the pore size of
membranes in MBR. This characteristic, coupled with low compressibility of the granular
sludge, can lead to a significant decrease in membrane fouling. Thus, the main form of
membrane fouling caused by the sludge can be eliminated by replacing flocculent sludge
with granular sludge, and the permeability throughout the operation would be conditioned
only by the solutes and colloids present in the medium (MENG et al., 2009; WANG et
al., 2013).

Flux is also an important factor that influences membrane fouling. A flux lower
than 20 L.m-2.h-1 produced 75 times more water than a flux of 40 L.m-2.h-1 in a flat sheet
membrane module (JOHIR et al., 2012). Under high flux operations there is a faster

25
sludge deposition on the membrane surface and the gel layer is formed more compactly
than when operated with smaller fluxes (LI, XIUFEN et al., 2005). The granules have a
lower tendency for membrane fouling than flocculent sludge and the use of granular
aerobic sludge allows the operation of the reactor with a higher flux (LI, WEN WEI et
al., 2012; TAY et al., 2007; ZHOU et al. Al., 2007).

Studies with aerobic granular sludge in MBR are carried out in batch or short run
continuous flux trials (CORSINO, S. F. et al., 2016; WANG et al., 2013). Aerobic
granules lose their stability faster in a continuous flux reactor than in SBR, due to the
absence of selection pressure in the continuous flux reactors as the high settling velocity
(CORSINO, SF et al., 2016). In almost all studies, the granular aerobic sludge was
produced in an SBR and inoculated in an MBR (SAJJAD; KIM; KIM, 2016).

4. Aerobic granular sludge at high temperatures

There is still little known about the formation of granular aerobic sludge at high
temperatures. Detailed knowledge regarding the effects of high temperatures on aerobic
granulation is still limited, although some studies on granular aerobic sludge have been
carried out at high temperatures (AB HALIM et al., 2015).

Thermophilic aerobic effluent treatment can be recommended for high-

temperature wastewater. The advantages of this treatment include high biodegradation
rates, low sludge production, deactivation of pathogenic organisms, and elimination of
pre-treatment cooling requirements. However, solid-liquid separation is a problem and
results in high solid concentrations in effluents (GUVEN, 2004).

Aerobic biological wastewater treatment at thermophilic temperatures (about 55

°C) produces sludge with poor flocculation. However, granules were formed at 55 °C
with diameters between 1 and 7 mm (GUVEN, 2004) or with a diameter between 1.2 and
1.9 mm and granule resistance to disintegration was comparable to aerobic mesophilic
granules (ZITOMER et al., 2007). The species Anoxybacillus flavathermus and
Pseudoxanthomonas taiwanensis, which are alkaliphilic or alkalitolerant, were isolated
from these granules indicating that high alkalinity may favor thermophilic granule-
forming populations (ZITOMER et al., 2007).

The effect of temperature on the granulation process was evaluated in an SBR to

achieve partial nitrification. The granules showed no significant changes to their
morphological characteristics with a temperature between 28 and 37 °C. The diameter of

26
more than 80% of the granules was greater than 6 mm when the temperature was raised
to 39 °C, but the density decreased from 28 to 19 g.L-1 and the sludge volume index (SVI)
increased from 33 to 80 mL.g-1. The system rapidly destabilized and nitrification ceased
when the temperature reached 41 °C (LÓPEZ-PALAU et al., 2013). The increase in
temperature causes an increase in the size and ash content of the granules (AB HALIM
et al., 2015).

Biological phosphate removal was higher in the system at 50 °C than at 30 °C and

40 °C, with efficiencies of 72%, 68%, and 67%, respectively. On the other hand, the
highest ammonia removal efficiency (97.5%) was observed at 30 °C, against 94.6% and
94.4% at 40 °C and 50 °C, respectively (AB HALIM et al., 2015). Phosphorus removal
reduced to insignificant values when the system temperature containing granules formed
at 20 °C was raised to 35 °C (EBRAHIMI et al., 2010).

5. Full-scale aerobic granular sludge systems

Aerobic granular sludge was developed in the 1990s and used initially in lab-scale
systems. Granular sludge was applied by Royal-HaskoningDHV in the development of
Nereda® technology in the early 2000s that allowed the deployment of full scale
applications. Nereda® technology was initially adapted for industrial application and then
expanded for domestic sewage treatment. The Nereda® process uses an optimized
sequential batch reactor cycle where the 4 steps of a typical RBS cycle are reduced to 3
steps. The first stage consists of the simultaneous fill and draw, in which the wastewater
is pumped into the reactor at the same time as the effluent is drawn. The second stage
consists of the aeration phase, in which the biological degradation of the organic matter
occurs. The cycle is finalized with the sedimentation phase where the separation of clear
effluent from biological sludge occurs. The Nereda® process can achieve nutrient
removal. Nitrifying bacteria accumulate in the outer layer of the granules and produce
nitrate, which will be consumed in the denitrification inside the granules, where anoxic
conditions prevail (GIESEN; THOMPSON, 2013).

The first full scale installation was implemented in the city of Epe, in the
Netherlands, in 2011 (PRONK et al., 2015, STUBBÉ, 2016). The reactors were designed
for average daily flow of 8,000 m3.d-1 and a maximum flow of 36,000 m3.d-1 (GIESEN;
THOMPSON, 2013). A granular sludge blanket was formed after a starting period of 5
months and consisted of more than 80% granules with a diameter greater than 0.2 mm
and more than 60% greater than 1 mm. The quality required for the effluent (7 mg .L-1 of
27
N and 1 mg.L-1 of P) is easily achieved throughout the year. Energy consumption is 58%
lower than the conventional activated sludge process due mainly to the absence of pumps
for recycling sludge return, and mixers used in conventional nutrient removal plants. In
addition, the specific volume required for granular aerobic sludge systems is 33% lower
than the volume of conventional activated sludge plants (PRONK et al., 2015).

The technology used relies on a sequencing fed-batch process with a constant

working volume. This is possible due to simultaneous feeding and effluent discharge, that
relies on a plug-flow pattern for displacement of effluent from the reactor (bottom feed
and discard at the top of the reactor) (PRONK et al., 2015).

The Netherlands currently has other full-scale aerobic granular sludge treatment
plants, such as those in the cities of Utrecht and Garmerwolde. The Garmerwolde granular
aerobic sludge installation went into operation in June 2013 with a maximum flow of
4200 m3.h-1 (PRONK et al. 2015) and phosphate removal was frequently used until May
2015, after which time only occasional dosing was applied, when the effluent phosphate
levels exceeded requirements (STUBBÉ, 2016).

The Utrecht and Garmerwolde installations are operated with cycles of about 6
hours, one hour feeding with simultaneous effluent withdrawal, 15 minutes of excess
sludge discharge, 4 hours of aeration, and one hour for settling. The cycle is reduced to a
minimum of 3 hours during rainy weather conditions. At both treatment sites, the aerobic
granular sludge system is operated together with a conventional activated sludge
treatment (STUBBÉ, 2016).

The success of Nereda® technology has increased interest in using granular sludge
at full-scale wastewater treatment plants and even larger plants are being designed and
built, including a plant in the city of Dublin and one in Utrecht with populations of
2,400,000 and 500,000, respectively. There are more than 16 plants operational
worldwide, including Kingaroy in Australia with an equivalent population of 12,500
inhabitants, operating since May 2016, that has achieved excellent effluent results to date
(AQUATEC MAXCON, 2016).

28
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37
 CAPÍTULO 2

Stability and resistance of aerobic granular sludge in bioreactors after addition of

calcium

Abstract

Granulation is a gradual process that makes flocculent sludge granular through the

simultaneous densification and selection of aggregates via sedimentation. Aerobic

granulation was established for the treatment of high organic load wastewater containing

nitrogen, phosphorus, toxic substances, and xenobiotics. Damage to granule structure

over time of bioreactor operation is one of the severest barriers to the practical application

of aerobic granular sludge. The addition of calcium, magnesium, and other metal ions that

contain coagulation properties may increase aggregation rates and granular structure

stability. Four sequential batch reactors (SBR) fed with kraft pulp mill effluent were

operated and monitored. Three reactors contained aerobic granular sludge and one

operated with flocculent sludge. One granular sludge SBR received the addition of 100

mg.L-1 of Ca2+; the second received 200 mg.L-1 of Ca2+, and the third received no

intentional addition of calcium. Organic matter removal efficiency from the reactors and

the effect of calcium on the morphological characteristics of the granules formed were

evaluated. Chemical oxygen demand (COD) and biochemical oxygen demand (BOD)

removal efficiencies were similar among all SBR, i.e., 60% and 90%, respectively. The

addition of calcium did not affect granule size. The addition of 100 mg.L-1 of Ca2+

increased the uniformity and the mechanical resistance of the granules and also increased

granule settling velocity by approximately 36%.

Keywords: aerobic granular sludge, calcium, pulp mill effluent, sludge settling velocity

38
Introduction

Aerobic granulation is a promising technique for wastewater treatment and has

significant potential for organic matter and nutrient removal, including recalcitrant and
1,2
toxic effluents . Granulation can be initiated by the adsorption and bacterial adhesion

to inert materials and inorganic precipitates as well as by adhesion of microorganisms to

other cells through physicochemical interactions. Filamentous bacteria increase the

granulation process, forming structures that support the adhesion of new cells 3.

Microorganisms produced by cell lysis release biopolymers that are actively

transported during the granulation process. These extracellular polymeric substances

(EPS) are composed mainly of proteins, polysaccharides, humic acids, nucleic acids, and
4,5
lipids and form a matrix involving the microorganisms that facilitates their

aggregation. EPS interactions and their characteristics are crucial for bioflocculation

because they constitute a major part of the sludge mass 6.

Biofloc surfaces are often negatively charged due to the presence of functional

groups in EPS. Carboxyl groups of uronic acids are deprotonated at pH values usually

found in activated sludge systems and contribute to the negative charge of bioflocs.

Proteins rich in amino acids containing carboxyl groups, such as glutamic and aspartic

acids also contribute to the negative charge of the bioflocs 6.

In the presence of Ca2+, Mg2+ and Fe2+ cations, the ionic bonds between carboxyl

and phosphate groups are reduced and granulation is increased. Metallic cofactors

improve the action of enzymes in the metabolism of viable cells 3. Bivalent cations bind

to negative functional groups in EPS thus stabilizing the biopolymer microorganism

matrix, which favors aggregation. Addition of Ca2+ to aerobic granular sludge produced

EPS with levels of polysaccharides higher than those of proteins, while the addition of

Mg2+ increased the protein content in relation to polysaccharides. Granulation rate,

particle size, and sludge settleability were rapidly increased by Ca2+ addition, whereas

39
sludge dewaterability was significantly enhanced by increasing Mg2+ 7. These ions are

probably constituents of polysaccharides or extracellular proteins. Although monovalent

ions can also neutralize EPS functional groups, the extracellular polymers mainly bind to

bivalent ions forming more stable complexes. The formation of calcium carbonate and

the linkage of calcium to EPS and cells are non-specific processes controlled by the

calcium ion gradient in the aqueous phase and by the granules. The addition of calcium

or simply the replacement of sodium bicarbonate (NaHCO3) by calcium hydroxide

(Ca(OH)2) for the neutralization of pH in the bioreactor can improve the granulation rate
8–11
.

Calcium precipitates neutralize negatively charged cell surfaces and function as

an inert support for bacteria. This phenomenon is important for the initial adsorption and

initiation of the aggregation process. Thus, calcium deficiency or loss may decrease

resistance or cause granule disruption 3.

In the presence of phosphates, the addition of calcium does not induce granulation

or can even be detrimental to granule formation. Phosphates can form calcium precipitates

inside the granules, damaging the environment required for maintenance of the granular

structure and bacterial activity 3.

High concentrations of calcium may have a negative effect on the granulation

process. Most studies with concentrations between 100 and 200 mgCa+2.L-1, presented a

positive effect on granulation, whereas those above 300 mgCa+2.L-1 led to precipitate

formation and had a negative influence on granulation 3.

Added sodium and excess calcium can reduce floc properties by replacing bivalent

cations. The ratio between the sum of the monovalent cations (meq.L-1) divided by the

sum of the bivalent cations with values greater than two indicates deterioration of the floc

properties 6.

40
 The objective of this study was to evaluate the characteristics of aerobic granular

sludge in a system treating bleached kraft pulp mill effluent. Aerobic granular sludge

characteristics such as size, settling velocity, strength, and organic matter removal

efficiency and filtrability were evaluated with and without the addition of calcium

chloride (CaCl2).

Methodology

Biological treatment. Biological treatment was performed in four (R1, R2, R3 and R4)

sequential batch reactors (SBR) operated in parallel (Figure 1). Each system had an

aeration tank with a functional working volume of 1.6 L (height/diameter ratio of 4).

Reactors temperature was maintained at 35°C by controllers connected to electric heaters.

Fine bubble aerators were placed at the bottom of the reactors to supply oxygen to the

biomass. Dissolved oxygen (DO) was maintained above 2 mg.L-1. The duration of each

cycle was 12 h and the volume exchange ratio was 50% resulting in a 24-hour hydraulic

retention time (HRT). Influent pH was kept between 6.5 and 7.5 and nitrogen and

phosphorus were added in the proportion COD: N: P equal to 200: 5: 1. The systems were

fed with effluent from the pulp mill. The reactors were inoculated with biological sludge

collected in the recirculation stream of an activated sludge treatment plant of a kraft pulp

mill. Reactor R1 were operated with flocculent sludge and reactors R2, R3 and R4 were

operated with granular sludge. Two SBR received CaCl2 as a calcium source. The

addition of calcium (100 mg.L-1 and 200 mg.L-1, respectively) in R3 and R4 aimed to

evaluate the effect of this element on the aerobic granulation process.

41
Figure 1. Experimental plan

Physical, chemical, and biological analyses. Soluble COD, soluble BOD, pH and total

suspended solids in the treated effluents were analyzed following the standard methods
12
. Calcium was determined by atomic absorption spectroscopy according to the Tappi

standard T266 om-02 13.

The size and the circularity of the granules were determined 14 using the ImageJ
15
program with measurements after SBR stabilization, i.e., after mature granules were

observed. Average granule diameters were determined to evaluate whether addition of

calcium affected their size. Diameters of the granules were compared among Reactors

R2, R3, and R4. The distribution frequency of the granule diameters was evaluated

throughout the test period after steady-state was achieved.

The specific growth rate of the granules (μ) was calculated based on the kinetic

model 16.

The settling velocity and the mechanical strength (integrity coefficient) of the

granules were determined 17.

42
Results and discussion

Characterization of the industrial effluent. The characteristics of the industrial effluent

were determined (Table 1).

Table 1. Feeding effluent physical-chemical parameters, n = 12

Parameter Unit Results (mean ± standard deviation)

pH - 7.08 ± 0.25
BOD5 mg.L-1 500 ± 13
COD mg.L-1 1142 ± 140
BOD/COD - 0.44 ± 0.09
Electrical conductivity mS.cm-1 2.31 ± 0.26
TSS mg.L-1 44 ± 8
Sodium mg.L-1 622 ± 102
R1 and R2 R3 R4
Calcium mg.L-1
170 ± 7 283 ± 2 385 ± 20

Effluent pH remained neutral, electrical conductivity and sodium concentration

18,19
were typical of bleached kraft pulp mills . Total suspended solid levels were low

because they had been previously removed by primary sedimentation at the pulp mill

treatment plant.

BOD and COD were similar to those reported for typical bleached kraft pulp mill

effluents 18,19. The organic matter in the effluent originates from wood (lignin, extractives,

and carbohydrates) during the wood pulping process 20.

Calcium concentrations in R1 and R2 correspond to that of the industrial effluent

used without added calcium from an external source (Table 1). The higher calcium

concentration in R3 and R4 compared to R1 and R2 is due to the intentional addition of

calcium chloride in these SBR.

43
Organic matter removal. The removal of organic matter was evaluated for soluble COD

and soluble BOD5 (Table 2).

Similar organic matter removal efficiency was achieved in the four SBR. A COD

removal of 60% and a BOD5 of 90% were obtained, which agrees with reported activated

sludge systems treating effluents from pulp mills 20.

Table 2. Soluble COD and BOD5 removal (mean ± standard deviation), n = 12 (BOD), n

= 52 (COD).

COD (%) BOD5 (%)

Treatments
Reactor R1 (FS) 60 ± 7 91 ± 4
Reactor R2 (GS) 61 ± 6 90 ± 5
Reactor R3 (GS100) 64 ± 6 90 ± 5
Reactor R4 (GS200) 62 ± 6 91 ± 4
FS: flocculent sludge; GS: granular sludge; GS100: granular sludge with addiction of 100
mg.L-1 Ca2+; GS200: granular sludge with addiction of 200 mg.L-1 Ca2+.

The granular aerobic sludge in R2, R3, and R4 achieved similar effectiveness

compared to the flocculent sludge in R1. This agrees with the organic matter removal

obtained from SBR in the treatment of recycled paper mill effluent 2.

The addition of calcium to R3 and R4 did not reduce organic matter removal

efficiency. No negative effect or reduced microbial activity was observed, demonstrating

that the presence of calcium at the concentrations used was inert to biological degradation.

Nevertheless, over the long term, the addition of calcium has been reported to increase

removal efficiency of organic matter due to improved formation and stability of the

granules 21,22.

Physical characteristics of the granules. The physical characteristics of the granules

formed in R2, R3, and R4 were evaluated considering diameter size, settling velocity, and

mechanical strength (Table 3).

44
Table 3. Diameter size (mm), settling velocity (Sett vel) and integrity coefficient (Int

Coef) of the granules (mean ± standard deviation), n = 5 (settling velocity and integrity

coefficient), n = 18 (diameter)

Diameter (mm) Sett vel (m.h-1) Int Coef ×100

Treatments
Reactor R2 (GS) 11.31 ± 0.80 23.53 ± 6.90 16.97 ± 9.67
Reactor R3
11.16 ± 1.01 32.73 ± 6.25 0±0
(GS100)
Reactor R4
11.33 ± 0.98 37.34 ± 8.53 1.56 ± 2.21
(GS200)
FS: flocculent sludge; GS: granular sludge; GS100: granular sludge with addiction of 100

mg.L-1 Ca2+; GS200: granular sludge with addiction of 200 mg.L-1 Ca2+.

The addition of calcium did not affect the size of the granules that had average

diameters of 11.16 to 11.33 mm, similar in the three SBR R2, R3 and R4. The granules

in the three SBR had larger diameters than those observed by other authors with and

without the addition of bivalent Ca2+ and Mg2+ cations 3,7,23–27. The formation of granules

with diameters larger than 10 mm is greater than typical granules reported in the literature

that are found to have a range from 0.8 to 4 mm 28–32. The larger diameter of the granules

produced in the present study could be the result of the characteristics of the industrial

effluent that contains a significant portion of recalcitrant organic matter (low

biodegradability) and by the SBR operational conditions. Compared to small granules,

mass transfer and diffusion limitations of large granules are more severe, which can lead

to EPS consumption by the cell core of the granules that would weaken the granular

structure 33.

The diameter of the granules was greater than 9 mm in all SBR (Figure 2). Reactor

R2, without calcium addition, presented 38.9% of the granule diameters between 10 and

11 mm, 27.8%, between 11 and 12 mm, and 27.8% greater than 12 mm. In SBR R3 and

R4, with added calcium, granule diameters remained mostly (50% of the observations)

45
within the range of 11 to 12 mm, presenting a smaller size variation and indicating greater

stability. Approximately, 86% of the granules formed in a bioreactor with the addition of

Mn2+ had diameter size in a range of 2.5 to 3.5 mm, whereas less than 58% of those were

within the same range without the addition of Mn2+. Thus, the addition of Mn2+ promoted
34
a narrower range and a greater similarity of the particle sizes of the granules . The

addition of low concentrations (0-1.05 mM) of a chelating agent, nitrilotriacetate (NTA),

reduced the growth rate and homogeneity of the granule size. The results showed that

20% of the granules had diameter equal to 0.5 mm with the addition of 1.05 mM NTA,

while 60% had a 0,5 mm diameter in the reactor without NTA 35.

Figure 2. Frequency of the diameter of the granules produced in reactors R2, R3, and R4.

Granules produced in reactors R3 and R4 were more uniform and larger than those

in reactor R2, with no added calcium. The granules in reactor R3 were denser and more

compact than those from the other reactors (Figure 3). Visually, the granules of reactor

R2 had a more fragile appearance than those from reactors R3 and R4.

46
Figure 3. Uniformity and granule size of sludge produced in reactors R1, R2, R3, and

R4.

The average coefficient of integrity of the granules was 16.97; 0.00 and 1.56 in

reactors R2, R3, and R4, respectively (Table 3). The resistance of the granules in reactors

R3 and R4, with added calcium, was higher than that in reactor R2, considering that the
17
higher the integrity coefficient, the lower the physical strength of the granules . The

integrity coefficient of reactor R3 was zero in all the tests, indicating absence of rupture

and proving the importance of increasing the resistance of the granules in the presence of

calcium. These results is in agreement with the finding that resistance of the granules

increased in the presence of 100 mg.L-1 of Ca2+ due to calcium precipitation within the

granules and the increase in polysaccharide content, forming a resistant core 36.

Granular aerobic sludge with high mechanical strength can improve the

performance of membrane bioreactors (MBR). Aerobic granular sludge in MBR increases

47
membrane fouling due to the rupture of the granules undergoing transmembrane pressure
2
. Thus, the addition of calcium in MBR with aerobic granular sludge may reduce the

fouling caused by the granule disruption.

Separation efficiency of the granules from the treated effluent depends on their

sedimentation rate. The sedimentation rate in reactors R2, R3, and R4 was 23.53; 32.73,

and 37.34 m.h-1, respectively (Table 3) showing a 36% and 59% increase in the settling

velocity of the granules in reactors R3 and R4 with the addition of calcium compared to

that of reactor R2 without added calcium. Positive bivalent and trivalent ions can bind to

negatively charged cells to form microbial nuclei, improving settling and strength

properties 36.

Sedimentation rates between 24.2 and 36.4 m.h-1, similar to those in reactors R2,
37
R3 and R4 were reported for granules with diameters between 1.2 and 1.8 mm .

Velocities higher than 40 m.h-1 were reported in granules with added calcium or

magnesium 5, much greater than values reported for flocculent sludge, from 3 to 5 m.h-1
38
.

Specific growth rate of aggregates. Growth of granules was initially slow (lag phase),

followed by a more marked growth phase (log phase) until stabilizing at a steady state

condition. The particle diameter was initially 0.6 to 0.8 mm, gradually increasing to

values greater than 11 mm after equilibrium. These values allowed the calculation of the

growth rate, which presented higher values in reactor R2 than in reactors R3 and R4

(Table 4).

48
Table 4. Specific growth rate (μ) of the granules produced in reactors R2, R3, and R4

Treatments D0 (mm) D (mm) Deq (mm) µ (10-3.d-1)

Reactor R2 (GS) 0.61 1.26 11.31 2.09

Reactor R3
0.78 1.38 11.16 1.99
(GS100)
Reactor R4
0.75 1.23 11.33 1.55
(GS200)
Size of microbial aggregates at lag phase (D0), exponential (D) and at equilibrium
(Deq); GS: granular sludge; GS100: granular sludge with addiction of 100 mg.L-1 Ca2+;
GS200: granular sludge with addiction of 200 mg.L-1 Ca2+.

The lower growth rates of the granules observed in reactors R3 and R4 are

important and may be related to the presence of calcium. Because of the lower growth

rate, the mechanical strength of the granules was higher. High growth rates are related to

a rapid proliferation of microorganisms, increasing the granule size and deteriorating

granule structure and density 36. In other study, heterotrophic and autotrophic bacteria in

aerobic granules had specific growth rates of 3.18 and 1.52 d-1, respectively. The fast

growing heterotrophs contribute to an increase of EPS, which is important for the

maintenance of the integrity and stability of the structure of the matured granules 39. The

size of granules and their specific growth rates decreased from 2 to 0.45 mm and from

0.085 to 0.065 d -1, respectively, due to the increased ratio of nitrogen to COD (N/COD).

The N/COD ratio contributes to the microbiological selection in the granules and

increases the population of nitrifying bacteria that have a slower growth rate than that of

heterotrophic bacteria 16.

Extracellular polymeric substances (EPS). The amount of EPS produced by the sludge

in each reactor was determined and polysaccharide (PS) and protein (PN) content and the

PS/PN ratio were quantified (Table 7).

49
Table 7. Polysaccharides (PS), proteins (PN) and PS/PN ratio in extracellular polymeric

substances (mg g-1VSS) of reactors R1, R2, R3 and R4

Treatments PS PN PS/PN
Reactor R1 61 86 0.71
Reactor R2 105 179 0.59
Reactor R3 91 60 1.53
Reactor R4 80 70 1.14

The PS production in the systems with granular sludge in reactors R2, R3, and R4,

was higher than in reactor R1 with flocculent sludge (Table 7). A large amount of PS

facilitates adhesion between cells and strengthens the microbial structure by forming a

strong, thick polymer matrix. The greater the microbial activity, the higher the PS, which

increases initial granule resistance 40. The higher PS content and the PS/PN>1 ratio of the

granules formed in reactors R3 and R4 may have contributed to the higher sedimentation

rate of these granules in relation to the granules formed in reactor R2, since amount of

EPS, especially polysaccharides, showed a positive correlation with the sedimentability

of the sludge 41,42.

The amount of protein was higher in the reactor with granular sludge without

added calcium, in reactor R2. In addition, the specific growth rate of the granules was

higher in reactor R2 (Table 4). It has been previously been shown that the increase in

particle size is faster with high protein content 7.

The total amount of EPS (PS + PN) was higher in reactors R2, R3, and R4, with

granular sludge, than in reactor R1, with flocculent sludge. EPS influences granulation

by changing surface characteristics such as surface charge and hydrophobicity. A lower

43
surface charge and greater hydrophobicity favors cell aggregation . Reactor R2

produced about 90% more EPS (PS + PN) than the others and had a higher growth rate

of the granules (Table 4). A large amount of EPS facilitates adhesion between cells due

to the presence of functional groups such as hydroxyl, carboxyl and starch in the

50
molecules of the extracellular polymer substances, rapidly increasing the size of the

granules 7.

Conclusions

The addition of calcium improved the physical characteristics of aerobic granular sludge,

but did not affect the efficiency of removal of organic matter in the reactors. The addition

of 100 and 200 mg.L-1 of Ca2+ increased the settling velocity of the granules formed with

calcium addition and its resistance, decreasing the integrity coefficient by 36% and 59%,

respectively. The mean diameter of the granules was similar in all granular sludge

reactors, but varied less in reactors with added calcium. The higher settling velocity and

mechanical strength and the high organic matter removal efficiency achieved in the

reactor with 100 mg.L-1 of Ca2+ added guaranteed greater stability of the bleached kraft

pulp mill effluent through the treatment process using granular aerobic sludge.

Acknowledgements

We would like to thank the following Brazilian agencies “Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq)”, “Coordenação de Aperfeiçoamento

de Pessoal de Nível Superior (CAPES)” and “Fundação de Amparo à Pesquisa do Estado

de Minas Gerais (FAPEMIG)” for their financial support. We would also like to thank

Celulose Nipo-Brasileira S.A for their support and the supply of materials. Dr. Phillip

John Villani (The University of Melbourne, Australia) revised and corrected the English

language used in this manuscript.

51
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Figure Legends

Figure 1. Experimental plan

Figure 2. Frequency of the diameter of the granules produced in reactors R2, R3, and R4.

Figure 3. Uniformity and granule size of sludge produced in reactors R1, R2, R3, and
R4.

54
 CAPÍTULO 3
Identificação dos microrganismos favoráveis à formação de grânulos

aeróbios mesofílicos presentes no tratamento biológico de efluentes de fábrica de

papel

RESUMO
Os grânulos aeróbios são grandes e compactos agregados microbianos que podem ser
utilizados no tratamento de águas residuárias. A adoção de lodo aeróbio granular em
biorreatores para o tratamento de efluentes é ainda considerada uma inovação e tem sido
tema de diversas pesquisas recentes. No presente trabalho, dezenove isolados
microbianos, obtidos em estudo realizado anteriormente, provenientes de grânulos
aeróbios mesofílicos foram avaliados em testes de co-agregação e determinação das
substâncias poliméricas extracelulares (SPE). Buscou-se avaliar a relação entre a
quantidade de SPE produzida e a contribuição do isolado no processo de granulação. Os
resultados indicaram que seis isolados dos gêneros Staphylococcus, Agrobacterium,
Enterobacter e Rhodococcus foram considerados como efetivos para a produção e
estabilidade dos grânulos aeróbios mesofílicos. A ausência destes isolados provocou um
aumento da quantidade de carboidratos e uma redução da quantidade de ácidos húmicos
e da relação proteína/polissacarídeo (PN/PS). A relação PN/PS está relacionada com a
hidrofobicidade do lodo granular e a sua redução prejudicou a formação de grânulos.

1. INTRODUÇÃO
Novas tecnologias têm ampliado a eficiência e reduzido custos do tratamento de
efluentes. O desenvolvimento do tratamento com lodo aeróbio granular tem demonstrado
que o processo pode apresentar elevadas eficiências de remoção de matéria orgânica e
nutrientes. Comparado ao lodo floculento do sistema de lodos ativados convencional, o
maior tamanho, a estrutura compacta e a alta retenção de biomassa do lodo aeróbio
granular implicam em uma maior velocidade de sedimentação e filtrabilidade do lodo
granular, além de alta tolerância à cargas orgânicas e toxicidade (LI, YONGMEI et al.,
2014; MORAIS; SILVA; BORGES, 2016; TAY et al., 2002; TOH et al., 2003).
A produção e a ação das substâncias poliméricas extracelulares (SPE) são
importantes para determinar as características dos agregados microbianos nos processos
55
de tratamento de efluentes (TAY et al., 2002). São essenciais para a formação de
agregados microbianos, uma vez que as bactérias ficam dispersas em uma matriz de SPE.
Tais substâncias possuem função estrutural e protetora nos agregados e afetam a
porosidade, a densidade, a carga elétrica, a hidrofobicidade, a estabilidade mecânica, além
de outras propriedades dos biofilmes (LIU; LIU; TAY, 2004).
As SPE são macromoléculas orgânicas secretadas por bactérias sob certas
condições ambientais, compostas principalmente por proteínas, polissacarídeos, ácidos
húmicos, ácidos nucléicos e lipídios (GAO et al., 2011). A composição das SPE pode
afetar a formação e as características dos grânulos aeróbios. A compactação do lodo
granular é obtida tanto pelo aumento do teor de proteínas quanto de polissacarídeos dos
SPE (CAUDAN et al., 2014). A resistência dos grânulos depende das condições de
crescimento e do tipo de substrato. Grânulos mais densos são formados em consequência
do crescimento de microrganismos com baixa taxa de crescimento e devido a interações
específicas envolvendo as SPE da matriz do grânulo (CAUDAN et al., 2014).
Os grânulos aeróbios podem levar semanas para serem formados a partir de lodos
ativados floculentos. Similar a outros tratamentos, a formação de grânulos pode ser obtida
por meio da seleção de culturas microbianas. A inoculação de alguns tipos de
microrganismos ao biorreator pode contribuir para o aumento da velocidade de formação
e da resistência mecânica dos grânulos (ADAV, LEE E LAI, 2010; IVANOV et al., 2006;
WAN et al., 2015). A mistura de uma cultura pura com elevada capacidade auto-
agregação com o lodo de um sistema de lodos ativados, resultou na formação de grânulos
aeróbios com diâmetro médio de 446 µm em apenas 8 dias (IVANOV et al., 2006).
Bactérias do gênero Sphingomonas sp. estavam presentes em grande quantidade no início
da granulação em reatores em batelada sequenciais (RBS) alimentados com efluente
sintético e contribuíram para a manutenção da estrutura e função dos grânulos (WAN et
al., 2016).
Interações físicas entre bactérias agregadas facilitam interações metabólicas,
comunicação intercelular e trocas genéticas (MASZENAN, LIU E NG, 2011). Estudos
sugerem que as características físicas dos grânulos são explicadas pela diversidade das
comunidades microbianas e da composição das SPE associadas a estas comunidades
(CAUDAN et al., 2014).
Em um trabalho prévio, determinou-se os tipos de microrganismos encontrados
no lodo aeróbio granular que contribuiram para a agregação microbiana e aumento da
resistência mecânica dos grânulos. Dezenove linhagens foram isoladas de um lodo
granular aeróbio formado em um RBS que tratava efluente de fábrica de papel. A
56
formação dos granulos foi obtida a partir da seleção microbiana. Os testes de co-
agregação indicaram que alguns isolados (2, 7, 9, 13, e 25) melhoraram a formação dos
granulos, enquanto outros (10, 14, 18, e 26) inibiram a granulação (MORAIS et al., 2016).
O objetivo deste trabalho foi quantificar as substâncias poliméricas extracelulares
produzidas pelos consórcios entre os microrganismos isolados no trabalho de Morais et
al. (2016) envolvidos no processo de granulação aeróbia e verificar a relação de SPE com
a resistência e estabilidade dos grânulos. Os microrganismos presentes nos grânulos
foram identificados.

2. METODOLOGIA
2.1. OBTENÇÃO DOS GRÂNULOS AERÓBIOS E DOS ISOLADOS

Os grânulos aeróbios mesofílicos foram obtidos em um reator em batelada

sequencial (RBS), alimentado com efluente de fábrica de papel reciclado (MORAIS et
al., 2016).
Alíquotas de 5 mL de lodo granular foram submetidas a três etapas de
centrifugação a 650 g por 2 minutos. Após cada centrifugação, o sobrenadante foi
descartado e o sedimentado re-suspenso em solução salina 0,85%. Ao final da terceira
centrifugação as amostras foram submetidas a pulsos de ultra-som (20 kHz, 4 s) para a
desagregação do lodo, utilizando-se o Ultrasonic homogenizer (Cole Palmer Instrument
Company 4710 series, Chicago).
As amostras desagregadas por ultra-som foram centrifugadas (650 g, 2 min) e
retiradas alíquotas de 100 µL do sobrenadante para obtenção de diluições seriadas de 10-
1
a 10-9.
Amostras de 100 µL de cada diluição, foram inoculados em placas de Petri
contendo meio R2A sólido (0,5 g de caseína hidrolisada; 0,5 g de extrato de levedura; 0,5
g de peptona; 0,5 g de dextrose; 0,3 g de fosfato dipotássico; 0,024 g de sulfato de
magnésio; 0,3 g de piruvato de sódio, 15,0 g de ágar e 1000 mL de água destilada). As
placas foram mantidas a temperatura ambiente e, após o surgimento das colônias,
realizou-se a repicagem para placas de meio R2A sólido para obtenção de culturas puras.
Cada cultura pura foi inoculada em meio R2A líquido e estocada em ultra-freezer a -80
°C.

57
 2.2. INDICE DE CO-AGREGAÇÃO COM AUSÊNCIA DE UM ISOLADO

Foram realizados 20 ensaios de co-agregação a partir de consórcios dos isolados,

um contendo todos os isolados e outras 19 combinações faltando um dos isolados em cada
uma delas, para verificar a influência de cada isolado obtido na formação dos grânulos.
O consórcio com todos os isolados foi denominado controle e os demais foram
identificados por “-n” que se refere ao isolado ausente no consórcio. Por exemplo, o
consórcio “-1” significa a combinação de 18 isolados na ausência do isolado 1, e assim
por diante.
Cada isolado foi inoculado, inicialmente, em meio R2A líquido e incubado a 30 +
1 ºC, sob agitação (110 + 10 rpm), por 48 horas.
Foram inoculadas alíquotas das culturas líquidas de cada isolado com densidade
ótica (DO) a 600 nm igual a 1,00 mL-1 em erlenmeyers de 250 mL contendo 75 mL de
meio R2A líquido. Os erlenmeyers foram incubados a 30 + 1 ºC, sob agitação (110 + 10
rpm) e foram retiradas 6 amostras de 5 mL de cada erlenmeyer após 2, 4, 8, 24, 48 e 72
horas do início da incubação. As amostras foram submetidas à leitura de DO (600 nm) e
armazenadas para extração e análise das substâncias poliméricas extracelulares (SPE).

2.3. EXTRAÇÃO E ANÁLISE DAS SPE

Foi transferido 1 mL de cada amostra para microtubos (2,0 mL), centrifugados

(11200 g, 4 ºC, 15 min) e o sobrenadante foi armazenado em outro microtubo para
quantificação das SPE livres. Os pellets foram re-suspensos em 20 mL de tampão fosfato,
o pH foi corrigido para 11, por meio da adição de NaOH 1M, seguido de aquecimento em
banho-maria (80 ºC, 30 minutos). Após essa etapa de extração, as amostras foram
centrifugadas (11200 g, 4 ºC, 10 min) e o sobrenadante armazenado para posterior análise
das SPE ligadas (MCSWAIN et al., 2005).
A caracterização química das substâncias poliméricas extracelulares produzidas
foi realizada por meio das análises dos conteúdos de carboidratos, proteínas, ácidos
húmicos e carbono orgânico total (COT).

2.3.1. Quantificação de carboidratos

Utilizou-se o método de dosagem fenol-ácido sulfúrico, no qual foram
adicionados 0,25 mL de fenol (5%) e 1,25 mL de ácido sulfúrico em 0,5 mL de cada
amostra. Após 10 minutos, os tubos foram agitados e colocados em banho de gelo por 20

58
minutos. Foi utilizada como padrão a curva de calibração utilizando sacarose (0 - 1,0
mg.mL-1). As leituras das absorbâncias foram realizadas em espectrofotômetro a 490 nm
(ALBALASMEH; BERHE; GHEZZEHEI, 2013).

2.3.2. Quantificação de proteínas e ácidos húmicos

Utilizou-se o método de Lowry modificado (FRØLUND et al., 1996), no qual é
necessário o preparo de dois reagentes (A e B) para as reações. O reagente A foi produzido
pela adição de 3 soluções, na proporção de 1:1:100 (10- 35 g de CuSO4. L-1 de água; 20-
70 g de KNaC4H4O6.4H2O.L-1 de água e 3- 70 g de Na2CO3 .L-1 de NaOH 0,35N). O
reagente B foi produzido com composição semelhante à do reagente A exceto pela
substituição da solução de CuSO4 por água deionizada. Nas reações foi utilizado também
o reagente Folin Ciocalteau (2N) diluído 10 vezes.
Os reagentes foram adicionados na sequência Amostra:Reagente A:Reagente
Folin, na proporção 1:1:1. Agitou-se a mistura e após 30 minutos realizou-se as leituras
em espectrofotômetro a 750 nm. Realizou-se o mesmo procedimento com o Reagente B.
Foi utilizada como padrão uma curva de calibração de 0-1,0 mg.mL-1 de albumina.
Para a determinação das proteínas presentes utilizou-se a seguinte formula:
Abs proteínas= 1,25 . (AbsA – AbsB)

A determinação de ácidos húmicos presentes nas amostras foi estimada utilizando

a fórmula:
Abs ácidos húmicos = AbsB – (0,2 . Abs proteínas)

2.3.3. Determinação de Carbono Orgânico Total (COT)

O COT foi determinado pelo método da oxidação catalítica (XUAN et al., 2010)
por meio de um analizador de carbono (TOC-Vcph, Shimadzu®, Quioto).

2.4. IDENTIFICAÇÃO DOS ISOLADOS OBTIDOS NA FORMAÇÃO DOS

GRÂNULOS
2.4.1. Extração do DNA
Os isolados foram inoculados em microtubos (2,0 mL) contendo 1 mL de meio
TY líquido (5 g de triptona, 3 g de extrato de levedura, 0,9 g de cloreto de cálcio
dihidratado e 1000 mL de água destilada). Foi efetuada a extração do DNA, após 24 horas
de incubação, conforme metodologia descrita por Kennedy et al., (2008), com
modificações. Foi adicionado 1 mL do tampão de lise (EDTA 100 mM, Tris–HCL 100

59
mM, CTAB 2%, SDS 1% e NaCl 1,5 M) a cada cultura. A mistura reacional foi
homogeneizada em vortex e aquecida em banho-maria a 65 ℃ por 20 min (com inversão
dos tubos a cada 5 min). Em seguida, a mistura foi centrifugada por 20 min a 2800 g. A
fase aquosa foi transferida para outro microtubo (2,0 mL) e adicionou-se a solução
fenol:clorofórmio:álcool isoamílico (25:24:1) no mesmo volume do coletado e
homogenizou-se em vortex, por 1 min, seguido de centrifugação por 10 min a 2800 g. O
sobrenadante foi transferido para outro microtubo (2,0 mL) e adicionou-se 0,7 volume de
isopropanol (100%) e 0,1 volume de acetato de sódio 3 M, inverteu-se os tubos
suavemente (10 vezes) e incubou-se a -20 ºC durante aproximadamente 12 horas. Os
tubos foram centrifugados por 10 min a 2800 g, descartando o sobrenadante, seguido da
lavagem do pellet com álcool 70% e nova centrifugação, por duas vezes. O pellet foi
deixado secar e então re-suspenso em 40 µL de água ultrapura.

2.4.2. Amplificação de DNA

O DNA de cada isolado foi amplificado utilizando os primers 27F (5′-
AGAGTTTGATCMTGGCTCAG-3′) e 1525R (5′-AAGGAGGTGATCCAGCC-3′). A
mistura reacional do PCRcontinha 0,3 µL de Taq DNA polymerase (Invitrogen, Carlsbad,
CA, USA), 0,5 µl de cada primer (10 µM), 2,5 µL do tampão de reação 10X, 0,5 µL de
dNTP’s (10 mM), 1,5 µL MgCl2 (50 mM), 1 µL da amostra de DNA e 18,2 µL de água
ultrapura, por amostra. A amplificação foi realizada em um termociclador (Mastercycler
Personal; Eppendorf, Hamburg, Germany) sob as condições: 94°C por 5 min, seguido por
14 ciclos (94 °C por 30s, 65 °C por 40s e 72 °C por 1 min), mais 15 ciclos (94 °C por 30s,
50 °C por 40s e 72 °C por 1 min); e a etapa de extensão a 72°C por 7 min.
Após essa etapa, os amplicons foram sequenciados no ACTGene Laboratory
(Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre,
Brasil) em um ABI-Prism 3100 Genetic Analyzer com capilares de 50 cm e polímero
POP6 (Applied Biosystems, Foster City, CA, USA).
Os dados obtidos após o sequenciamento foram tratados através dos programas
MEGA (V. 6.0) e Bioedit, os contigues obtidos foram submetidos ao BLAST (NCBI,
2015) para o processo de identificação dos isolados.

60
3. RESULTADOS E DISCUSSÃO
3.1. ENSAIO DE CO-AGREGAÇÃO - DENSIDADE ÓTICA DOS
CONSÓRCIOS

O crescimento das células nos consórcios foi monitorado pela densidade ótica
durante 72 horas. Os consórcios apresentaram alterações mínimas na densidade ótica
(Figura 1) após 24 horas, evidenciando a fase estacionária e indicando que, durante esse
período, houve a produção/liberação das SPE que favorecem a formação de grânulos.

2 Horas 4 Horas 8 Horas 24 Horas 48 Horas 72 Horas

1,8
DENSIDADE ÓPTICA (600 NM)

1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
-1 -2 -3 -4 -5 -7 -8 -9 -10 -11 -13 -14 -15 -18 -19 -23 -24 -25 -26 T
CONSÓRCIOS

Figura 1. Densidade óptica (600 nm) das amostras coletadas de cada consórcio, em
diferentes intervalos de tempo (horas), durante experimento de co-agregação (-
n=consórcio com ausência do n-ésimo isolado, T = consórcio controle com todos os
isolados).

Os valores de densidade ótica (DO) foram similares ao longo do tempo entre os

consórcios. Houve um aumento gradual da DO ao longo do monitoramento até sua
estabilização em 1,5, aproximadamente, após 24 horas (Figura 1). Dessa forma, a
ausência de um dos isolados não afetou o crescimento microbiano dos consórcios.
A medição da densidade ótica é um método rápido e confiável, mas baseia-se na
suposição de que as bactérias crescem como células únicas de igual tamanho e que as
células são dispersas uniformemente na cultura líquida (HAABER et al., 2016). No caso
de culturas formando aglomerados de células com uma estrutura em três dimensões, a

61
correlação entre os dados de densidade óptica e o número de células deve ser verificada,
uma vez que parte da biomassa pode cobrir camadas internas de células (DE
CARVALHO et al., 2005). Outros desafios que podem potencialmente afetar a medição
da densidade óptica incluem células viáveis mas não cultiváveis ou células não viáveis
mas intactas presentes na cultura, bem como células no processo de divisão (HAABER
et al., 2016).
Em um biorreator, a densidade ótica pode se correlacionar com a concentração de
sólidos suspensos voláteis no tanque de aeração (SSVTA). A DO acompanhou as
variações dos SSVTA durante o processo de formação de grânulos aeróbios em um RBS
para o tratamento de 2,4-diclorofenol (KHAN; KHAN; SABIR, 2011). Além disso, a
presença de substâncias tóxicas inibe o crescimento e reduz a DO. A adição de 2 mg.L-1
e 3mg.L-1 de Cr6+ provocou uma redução na DO de 1,0 para 0,8 e 0,3, respectivamente
(YANG et al., 2016), enquanto que a adição de 3,5 mM de dodecil sulfato de sódio causou
uma diminuição da DO de 1 para 0,2 em apenas 30 minutos de incubação
(KLEBENSBERGER et al., 2006).

3.2. DETERMINAÇÃO DAS SPE

3.2.1. Quantificação de carboidratos
Os consórcios com ausência dos isolados que prejudicam a granulação (-4, -10, -
14, -18 e -26) apresentaram, de maneira geral, uma redução da produção de carboidratos
após 48 h, enquanto os consórcios com ausência dos isolados que contribuem para a
formação de agregados (-2, -7, -9, -13, -19 e -25) mantiveram ou aumentaram a produção
de carboidratos após 48 h levando a uma maior quantidade de carboidratos em relação ao
controle (Figura 2). Além disso, o consórcio controle (T) apresentou baixa produção de
carboidratos em comparação aos demais, 0,3 mg.mL-1 e 0,16 mg.mL-1 nas medições
realizadas em 48 h e 72 h, respectivamente.
Os carboidratos são componentes hidrofílicos dos SPE e o excesso de carboidratos
(principalmente se aliado à baixa relação proteína/carboidrato) resulta em um aumento
do potencial zeta, prejudicando a formação de grânulos (TU et al., 2010). Dessa forma,
os isolados indicados como prejudiciais ao processo de granulação podem ter reduzido a
formação de agregados pela sua maior produção de carboidratos.

62
 48 Horas 72 Horas

Carboidratos (mg.mL-1)
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
-1 -2 -3 -4 -5 -7 -8 -9 -10-11-13-14-15-18-19-23-24-25-26 T
Consórcios

Figura 2. Quantificação de Carboidratos (mg.mL-1) nas substâncias poliméricas

extracelulares extraídas das amostras coletadas, em diferentes intervalos de tempo
(horas), durante experimento de co-agregação (-n=consórcio com ausência do n-ésimo
isolado).

De acordo com Seviour et al., (2012), os grânulos aeróbios contêm uma

comunidade microbiana multiespecífica, que sintetizam uma grande diversidade de
exopolissacarídeos desempenhando diferentes papéis estruturais e distinguem-se dos
flocos do processo de lodos ativados convencional pela presença de exopolissacarídeos
gelatinosos.
A sedimentabilidade do lodo granular está diretamente relacionada com a
quantidade de SPE produzida (KIM et al., 2014). Os polissacarídeos são importantes na
etapa de formação dos grânulos, mas a estabilidade dos mesmos depende do núcleo
proteico (ZHU et al., 2015). A quantidade de SPE depende, entre outros fatores, da
comunidade microbiana presente no grânulo e das condições de operação do reator. A
diminuição do tempo de sedimentação do lodo provocou o arraste dos microrganismos
de sedimentação lenta e o aumento a comunidade microbiana com boa floculação, o que
resultou no aumento rápido dos teores de proteína (PN) e polissacarídeos (PS). No
entanto, com a maturação do lodo granular, o teor de PS diminuiu devido ao consumo
dos polissacarídeos pelos microrganismos durante a fase de inanição (ZHU et al., 2012).

3.2.2. Quantificação de Proteínas

A produção de proteínas dos consórcios variou entre 0,08 mg.mL-1 e 0,35 mg.mL-

63
1
e não houve um comportamento regular entre os consórcios na ausência dos isolados
que beneficiam ou entre os que prejudicam a formação de agregados (Figura 3). A
produção de proteínas nos consórcios -2, -4, -8, -23, -25, -26 e T aumentou entre 48 h e
72 h, indicando que a ausência destes isolados foi benéfica à formação de agregados.

48 72
0,4
Proteínas (mg.mL-1)

0,3

0,2

0,1

0
-1 -2 -3 -4 -5 -7 -8 -9 -10-11-13-14-15-18-19-23-24-25-26 T
Consórcios

Figura 3. Quantificação de Proteínas (mg.mL-1) nas substâncias poliméricas

extracelulares extraídas das amostras coletadas, em diferentes intervalos de tempo
(horas), durante experimento de co-agregação (-n=consórcio com ausência do n-ésimo
isolado).

As proteínas são constituintes hidrofóbicos dos SPE e contribuem para a

diminuição do potencial zeta, favorecendo a formação, além de aumentarem a
estabilidade estrutural dos grânulos (DI LACONI; RAMADORI; LOPEZ, 2006;
MCSWAIN et al., 2005; TU et al., 2010). Zhu et al. (2015) demonstrou que os teores de
proteínas de lodos granulares aeróbios e anaeróbios foram maiores do que os de lodos
floculentos, apresentando uma correlação linear negativa com a carga superficial do lodo
e favorecendo a formação de grânulos.
A falta de uma tendência de aumento ou diminuição da produção de proteínas
pelos consórcios dificulta a avaliação da influência deste parâmetro na formação dos
agregados. Pode-se avaliar também a relação entre o conteúdo de substâncias
nitrogenadas (PN) e polissacarídicas (PS). A relação PN/PS pode influenciar
positivamente a hidrofobicidade e o potencial zeta, contribuindo para o aumento da
habilidade de agregação (ZHANG et al., 2015). Além disso, grânulos formados com

64
relação PN/PS menor do que 1 são suscetíveis a desagregação, uma vez que as interações
célula-célula são fracas (JIANG; TAY; TAY, 2002; LIU; LIU; TAY, 2004; ZHANG et
al., 2015).
Os consórcios -4, -10, -14, -18 e -26, nos quais estavam ausentes os isolados
prejudiciais à granulação, apresentaram, com exceção do -26, relação PN/PS ≥ 1 (Tabela
1). Uma maior relação PN/PS contribui para uma maior agregação e maior estabilidade
dos agregados (ZHANG et al., 2015).

Tabela 1. Relação PN/PS dos consórcios de isolados

Consórcio PN/PS Consórcio PN/PS

-1 1,79 -13 0,72

-2 0,88 -14 2,38

-3 0,92 -15 1,08

-4 7,14 -18 1,00

-5 0,79 -19 2,56

-7 1,25 -23 1,56

-8 5,56 -24 1,96

-9 1,15 -25 8,33

-10 1,79 -26 0,78

-11 3,45 T 3,25

A relação PN/PS dos grânulos é maior do que a dos flocos (SAJJAD; KIM, 2015;
ZHU et al., 2015). Grânulos aeróbios apresentaram maior teor de proteínas (PN/PS entre
1,4 e 1,6) enquanto flocos apresentaram maior teor de polissacarídeos (PN/PS = 0,5)
(BASUVARAJ; FEIN; LISS, 2015). As PN possuem ainda maior afinidade por cátions
como o Ca2+ e o Mn2+ e facilitam a ligação das SPE com estes íons, reduzindo a carga
superficial e promovendo a adesão celular (ZHU et al., 2015).

3.2.3. Quantificação de ácidos húmicos

A produção de ácidos húmicos (AH) variou entre 0,13 mg.mL-1 e 0,53 mg.mL-1
(Figura 4).

65
 48 72
0,6

0,5
Ácidos húmicos (mg.mL-1)
0,4

0,3

0,2

0,1

0
-1 -2 -3 -4 -5 -7 -8 -9 -10 -11 -13 -14 -15 -18 -19 -23 -24 -25 -26 T
Consórcios
Figura 4. Quantificação de ácidos húmicos (mg.mL-1) nas substâncias poliméricas
extracelulares, em diferentes intervalos de tempo (horas), durante experimento de co-
agregação (-n=consórcio com ausência do n-ésimo isolado).

Dentre os consórcios com ausência dos isolados favoráveis à granulação (-2, -7, -
9, -13, -19 e -25), os consórcios -2, -9, -13 e -25 apresentaram menor concentração de
ácidos húmicos do que o controle em 72 h, o que indica que a ausência destes isolados
pode ser prejudicial à formação de grânulos pela redução da quantidade de AH
produzidos. Por outro lado, entre os consórcios com ausência dos isolados prejudiciais à
granulação (-4, -10, -14, -18 e -26), os consórcios -4, -14 e -18 produziram maior
quantidade de ácidos húmicos do que o controle em 72 h indicando um favorecimento da
produção de AH com a ausência destes isolados.
Os ácidos húmicos estão relacionados entre as substâncias poliméricas produzidas
durante a formação dos grânulos aeróbios e atuam na estrutura dos grânulos (GAO et al.,
2011). Os grânulos aeróbios podem ser considerados um biossorvente eficaz para o
tratamento de metais pesados e os ácidos húmicos desempenham um papel importante na
sorção destes íons no lodo aeróbio granular (WEI et al., 2016). Wang et al. (2014)
observou que o aumento da concentração de Cr6+ até 30 mg.L-1 resultou em um aumento
do teor de substâncias proteicas e ácidos húmicos do lodo aeróbio granular. Além disso,
os ácidos húmicos contribuem também para a remoção de compostos tóxicos. O teor de
ácidos húmicos aumentou de 134,1 mg.L-1 para 138,4 mg.L-1 após a exposição do lodo a
20 mg.L-1 de 2,6-diclorofenol (LI, KAI et al., 2016).
A concentração de ácidos húmicos pode sofrer pouca influência das condições

66
operação do reator. A variação da relação DQO/N (igual a 1, 2 e 4) não afetou a
quantidade de proteína, ácido fúlvico e ácido húmico do lodo (LUO et al., 2014). O lodo
alimentado com maior relação alimento / microrganismo (A/M) melhorou o processo de
granulação e produziu mais polissacarídeos (PS) e proteínas (PN) do que o lodo com
menor carga de DQO, enquanto as substâncias húmicas e os ácidos urônicos não
apresentaram variação (KIM et al., 2014). O aumento da quantidade de ácido húmico e
fúlvico pode ser resultante da decomposição de células mortas e de compostos orgânicos
macromoleculares, como PN e PS (WANG et al., 2014).

3.3. IDENTIFICAÇÃO DOS ISOLADOS OBTIDOS

Os gêneros de todos os isolados obtidos a partir dos grânulos aeróbios foram

identificados (Tabela 2).

Tabela 2. Identificação dos isolados obtidos a partir dos grânulos aeróbios mesofílicos
formados durante o tratamento de efluentes de indústria de papel reciclado.
Gênero Isolados
Acinetobacter 1, 4, 5, 10, 15, 18 e 23
Agrobacterium 2, 13 e 19
------------- 3
Enterobacter 7, 8, 11, 14, 24 e 26
Staphylococcus 9
Rhodococcus 25

Os isolados favoráveis à formação dos grânulos (2, 7, 9, 13, 19 e 25) pertencem a

quatro dos cinco gêneros identificados (Tabela 3). Bactérias desses quatro gêneros são
descritas como produtoras de SPE com características que favorecem a formação de
biofilmes e grânulos (LV et al., 2014).

67
Tabela 3. Identificação dos isolados favoráveis à formação dos grânulos aeróbios
obtidos.
Isolado Identificação
2 Agrobacterium sp.
7 Enterobacter sp.
9 Staphylococcus sp.
13 Agrobacterium sp.
19 Agrobacterium sp.
25 Rhodococcus sp.

Espécies de Enterobacter foram relatadas como produtoras de grande quantidade

de polissacarídeos e SPE de adesão intracelular, os quais são substâncias primárias
envolvidas diretamente na formação de biofilmes e grânulos (LIMOLI; JONES;
WOZNIAK, 2015; TORRES et al., 2012). A curva de crescimento de Enterobacter
aerogenes mostrou que a secreção dos SPE era paralela ao crescimento celular, com a
máxima liberação de SPE observada na fase estacionária inicial (SALEHIZADEH; YAN,
2014).
Isolados de Agrobacterium sp. foram observados na superfície de grânulos.e
geraram grande quantidade de proteína e floculantes. Além disso, foi avaliada a utilização
de fósforo na produção de SPE de espécie de Agrobacterium e observou-se que limitação
de fósforo aumenta a produção das substâncias que auxiliam na formação de biofilmes e
grânulos por espécies desse gênero bacteriano (HUANG et al., 2012).
O Rhodobacter é um gênero bacteriano desnitrificante anaeróbio que também é
capaz de secretar SPE para auxiliar a fixação. Estas espécies devem contribuir para a
formação de grânulos aeróbios com a sua SPE segregada (LV et al., 2014). As SPE
produzidas por espécies de Rhodococcus apresentam propriedades mecânicas, devido a
formação de ligações não espécíficas. Além disso, algumas espécies têm sido amplamente
utilizadas em processos de biorremediação para reduzir contaminantes na água e solo
(PEN et al., 2015; RODRIGUES et al., 2006).
Exopolissacarídeos catiônicos como a adesina intercelular e poli-N-
acetilglicosamina, encontradas em biofilmes de Staphylococcus epidermidis e
Staphylococcus aureus, respectivamente, desempenham importante papel estrutural em
biofilmes (SEVIOUR et al., 2012). O Staphylococcus aureus é um patógeno humano que
68
provoca uma grande quantidade de doenças que vão desde infecções de pele benignas até
condições fatais, como bacteremia, endocardite infecciosa e infecções crônicas
(HAABER et al., 2016). A patogenicidade do S. aureus está relacionada com a sua
elevada capacidade de auto-agregação e de formar biofilmes. Além disso, S. aureus
agrega-se eficientemente no plasma humano, uma condição que está ligada à virulência,
e pode formar agregados planctônicos que protegem as células contra antibióticos
(HAABER et al., 2016).
Os isolados prejudiciais à formação dos grânulos (4, 10, 14, 18 e 26) são espécies
dos gêneros Acinetobacter e Enterobacter.
O gênero Acinetobacter possui membrana celular altamente hidrofóbica e pode
produzir excesso de SPE, possuindo alta capacidade de auto-agregação e capacidade de
aderência a superfície sólida (LV et al., 2014; PHUONG; KAKII; NIKATA, 2009).
Interações hidrofóbicas são importantes para a co-agregação de isolados de Acinetobacter
sp. e outras bactérias constituintes do lodo (PHUONG; KAKII; NIKATA, 2009).
Além disso, bactérias do gênero Acinetobacter possuem características que estão
relacionadas com o aumento da resistência elevada degradação de substâncias tóxicas.
Isolados de Acinetobacter calcoaceticus apresentaram autoagregação e elevada remoção
de fenol, formando grânulos estáveis com diâmetros de 2,3mm. Os índices de
autoagregação das Acinetobacter calcoaceticus tiveram correlação positiva com a
quantidade de proteínas extraídas dos agregados (ADAV; LEE, 2008). Substâncias
produzidas por espécies do gênero Acinetobacter apresentam polissacarídeos capsulares,
os quais conferem vantagens de proteção para as células. Biofilmes de Acinetobacter
baumannii protegeram as células contra exposição à tobramicina (DAVENPORT; CALL;
BEYENAL, 2014).

4. CONCLUSÕES
Foram obtidos 19 isolados cultiváveis a partir dos grânulos aeróbios mesofílicos
produzidos durante o tratamento de efluente de uma fábrica de papel. Com base nos
resultados das SPE, constatou-se que há diferenças na efetividade da influência de cada
isolado na formação dos grânulos. Os isolados 2, 7, 9, 13, 19 e 25 foram avaliados como
favoráveis para a formação e estabilidade dos grânulos aeróbios mesofílicos e
apresentaram, de maneira geral, maior produção de carboidratos, proteínas e relação
PN/PS superior à 1, o que contribui para o processo de granulação aeróbia.
Esses isolados benéficos à grânulos aeróbios foram identificados como

69
pertencentes aos gêneros Agrobacterium, Enterobacter, Staphylococcus e Rhodococcus,
que produzem grande quantidade de SPE auxiliando na formação e manutenção de
biofilmes e grânulos.

5. AGRADECIMENTOS
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e Fundação de
Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) pelo apoio financeiro.

70
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 II. Conclusões e recomendações gerais
O lodo aeróbio granular apresenta grandes vantagens em comparação a outros
processos de tratamento biológico. As características físicas do lodo garantem uma
melhor clarificação do efluente tratado, seja por sedimentação em um decantador
secundário ou por filtração por membranas. Além disso, os grânulos aeróbios podem ser
aplicados na remoção de nutrientes e substâncias tóxicas, apresentando elevadas
eficiências. Tais vantagens contribuem para o aumento do número pesquisas visando uma
maior compreensão do processo de granulação aeróbia. A revisão de literatura,
apresentada no Capítulo 1, indicou os principais parâmetros que influenciam na formação
do lodo aeróbio granular e mostrou que sua aplicação em plantas de larga escala ainda se
restringe a um número reduzido de estações de tratamento de águas residuárias em todo
o mundo, mas é previsto um aumento da implantação de ETE em larga escala com lodo
granular.

A aplicação de 100 mg.L-1 aumentou a velocidade de sedimentação e a resistência

dos grânulos formados no RBS com efluente de fábrica de polpa celulósica. No entanto,
o efeito de cátions divalentes pode variar em função da presença de agentes quelantes no
reator. Assim, a avaliação da presença de agentes quelantes no efluente de polpa
celulósica ou a adição destes no reator acrescentaria informações importantes para a
melhoria da granulação aeróbia.

Dezenove isolados obtidos a partir de lodo aeróbio granular em RBS tratando

efluente de fábrica de papel foram identificados. Isolados dos gêneros Agrobacterium,
Enterobacter, Staphylococcus e Rhodococcus favoreceram a formação e estabilidade dos
grânulos e produziram grande quantidade de SPE.

Os efluentes gerados no processo de polpação kraft apresentam temperaturas em

torno de 55 °C e precisam ser resfriados antes da estação de tratamento de efluentes por
lodos ativados convencional. A adaptação do lodo granular a temperaturas termofílicas
reduziria o custo de resfriamento do efluente e favoreceria o reuso do efluente tratado no
processo produtivo. No entanto, o aumento da temperatura dos sistemas com lodo
granular leva à redução da resistência e desintegração dos grânulos. O conhecimento
detalhado sobre os efeitos da temperatura sobre a granulação aeróbica ainda é limitado,
embora alguns estudos sobre o lodo aeróbio granular tenham sido realizados a altas
temperaturas.

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 O efeito da adição de cálcio na filtrabilidade do lodo aeróbio granular poderia ser
tema de um estudo posterior. O diâmetro dos grânulos e o aumento da resistência
mecânica provocado pela adição de 100 mg.L-1 de Ca2+ pode reduzir a incrustação dos
módulos de membranas e melhorar o funcionamento de um BRM.

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ANEXOS

78
 Determinação do tamanho médio dos grânulos

A determinação do tamanho e circularidade dos grânulos foi realizada por meio

do uso do programa ImageJ (RASBAND, 1997). Os diâmetros foram calculados como
diâmetros circulares equivalentes (LOCHMATTER; GONZALEZ-GIL; HOLLIGER,
2013).

Determinação da taxa específica de crescimento dos grânulos

A taxa específica de crescimento dos grânulos (�) foi calculada com base no
modelo cinético desenvolvido por Yang et al., (2004):

R – R0 = (Req – R0)[1 - ℮-µ(t-t0)]

Onde,
R = tamanho dos agregados no tempo t (mm)
Req = tamanho dos agregados no equilíbrio (mm)
µ = taxa específica de crescimento dos agregados (dia-1)
t0 = tempo no final da fase lag
R0 = tamanho dos agregados microbianos no tempo t0.

Na determinação da taxa específica, o valor de R utilizado refere-se ao

diâmetro dos grânulos 30 dias após o final da fase lag. Para este valor de tempo (t = 30)
todos os sistemas estavam em fase de crescimento dos grânulos e não tinham atingido o
estado estacionário.

Determinação da velocidade média de sedimentação

Para determinação da velocidade média de sedimentação, foi utilizado o

procedimento proposto por Ghangrekar; Asolekar; Joshi, (2005). Tal procedimento se
baseia na utilização de uma coluna de diâmetro igual a 7,5 cm e altura igual a 75 cm
(coluna de sedimentação) preenchida com água de torneira (Figura 1).

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 Figura 1 - Colunas utilizadas para realização do teste de velocidade de sedimentação dos
grânulos aeróbios.

Em cada coluna, foi acrescentado cerca de 25 mL de lodo diluído (5 – 10 vezes).

Água de torneira foi utilizada para a determinação da velocidade de sedimentação, pois o
pH e a força iônica da água de torneira é adequada para esta finalidade, e o tamanho dos
grânulos é suficiente para permitir que estes não sejam afetados pelo estresse osmótico.
A quantidade de lodo sedimentado ao fundo da coluna foi coletada em intervalos de
tempo fixos (0,5; 1; 1,5; 3; 7,5; 15 e 60 minutos). Determinou-se o SST de cada amostra,
para analisar a fração de lodo sedimentado em cada instante. A velocidade média de
sedimentação foi calculada pela equação:

∑ ���
��é .� ��. =
�

Onde,
vméd.sedim. = velocidade média de sedimentação
m = massa da fração de lodo sedimentada;
v = velocidade de sedimentação da fração;
M = massa total da amostra de lodo.

Determinação da resistência dos grânulos

O procedimento utilizado por Ghangrekar; Asolekar; Joshi, (2005) foi utilizado

para a determinação da resistência dos grânulos. O teste foi baseado na premissa de que
80
se os grânulos são submetidos à tensão de cisalhamento do fluido, a quantidade de lodo
liberado no fluido (pelo desprendimento dos grânulos) será função da resistência dos
grânulos. Uma amostra de lodo do reator foi diluída 10 vezes com água de torneira. Para
separar os grânulos do lodo floculento, a amostra de lodo diluída (25 mL) foi deixada
sedimentar na coluna de sedimentação (Figura 1). A fração de grânulos que sedimentaram
em 1 minuto foi utilizada para a determinação da resistência. A força de cisalhamento foi
introduzida, ainda que indiretamente, submetendo estes grânulos sedimentados em
frascos cônicos contendo água de torneira em um volume total de 150 mL, os quais foram
levados à um agitador (“shaker”). Cada amostra foi submetida ao mesmo grau de agitação
(200 rpm por 5 minutos). Após a agitação a amostra foi mantida em repouso durante 1
minuto. Foi realizada a análise de sólidos suspensos totais do sobrenadante e do
decantado. Os resultados das análises de SST foram utilizados para a determinação do
coeficiente de integridade definido como a razão entre a massa de sólidos do sobrenadante
dividida pela massa total de sólidos da amostra, ou seja:

ó�
� � � � =
ó�

Quanto maior a resistência dos grânulos, menor será a massa de sólidos no

sobrenadante, visto que esta é resultado do rompimento dos grânulos pela agitação. Dessa
forma, quanto menor o coeficiente de integridade de uma amostra, maior a resistência dos
grânulos. Assim, um coeficiente de integridade igual a 1 indicaria a completa ruptura dos
grânulos, indicando baixa resistência, e um coeficiente de integridade igual a 0 indicaria
que não houve ruptura dos grânulos, indicando elevada resistência.

Observações microscópicas

As observações microscópicas foram realizadas, diariamente, com amostras do

lodo fresco, utilizando um microscópio ótico com contraste de fase, da marca LEICA,
modelo DMLS. Foi acompanhado o desenvolvimento dos grânulos e observado as
características morfológicas do lodo biológico durante o processo de granulação. Foi
utilizada uma máquina digital, da marca Nikon, modelo COOLPIX 4500 para obtenção
de fotomicrografias dos grânulos e das características morfológicas do lodo biológico.

Purificação dos amplicons

Os amplicons foram purificados e submetidos a gel de agarose (1%) (Figura 2).

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Figura 2. Gel de agarose (1%) das amostras de DNA, amplificadas com os primers 27F e 1525R (M=
Marcador)

82